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Coordination of MRNA 3' end formation and nuclear export by a nuclear poly(A)-Binding protein

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Title:
Coordination of MRNA 3' end formation and nuclear export by a nuclear poly(A)-Binding protein
Creator:
Nykamp, Keith Robert, 1974-
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Language:
English
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viii, 167 leaves : ill. ; 29 cm.

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Subjects / Keywords:
DNA ( jstor )
Exons ( jstor )
Heterogeneous nuclear ribonucleoproteins ( jstor )
In vitro fertilization ( jstor )
Messenger RNA ( jstor )
Polyadenylation ( jstor )
Proteins ( jstor )
RNA ( jstor )
Splicing ( jstor )
Yeasts ( jstor )
Department of Molecular Genetics and Microbiology thesis Ph.D ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Molecular Genetics and Microbiology -- UF ( mesh )
Heterogeneous-Nuclear Ribonucleoproteins -- genetics ( mesh )
Polyadenylation -- genetics ( mesh )
RNA Polymerase II -- genetics ( mesh )
RNA, Messenger -- genetics ( mesh )
RNA-Binding Proteins -- genetics ( mesh )
Research ( mesh )
Yeasts ( mesh )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D)--University of Florida, 2003.
Bibliography:
Bibliography: leaves 138-166.
Additional Physical Form:
Also available online.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Keith Robert Nykamp.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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79277005 ( OCLC )

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COORDINATION OF MRNA 3' END FORMATION AND NUCLEAR EXPORT
BY A NUCLEAR POLY(A)-BINDING PROTEIN












By

KEITH ROBERT NYKAMP

















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2003






























Dedicated to my grandparents, parents and brothers,
my wife, Dawn, and son, Jonas.














ACKNOWLEDGEMENTS

I would like to thank my mentor, Maurice Swanson, for providing me with the resources and intellectual support necessary to complete my doctoral dissertation. During my graduate studies, he has challenged me to defend my hypotheses with unequivocal data and has equipped me with the tools needed for a lifetime of scientific inquiry. I also thank my committee members, Alfred Lewin, Stephen Sugrue, and Thomas Yang, for their valuable assistance at important times throughout my graduate career. I extend a special thanks to James Dahlberg, from the University of Wisconsin-Madison, for taking time out of his hectic schedule to serve as my outside examiner in Gainesville. Importantly, I thank past and present members of the Swanson Lab, in particular James Anderson, Ron Hector, and Carl Urbinati, for their help with many of the experiments described in this manuscript. I would also like to thank Lionel Minvielle-Sebastia for providing the in vitro polyadenylation results and John Aitchison for identifying purified proteins by mass spectrometry. Finally, my wife, Dawn, deserves special mention because without her love and support none of this would have been possible.














TABLE OF CONTENTS

ACKNOW LEDGEMENTS ........................................................................ iii

A B S T R A C T .......................................................................................... vii

INTRODUCTION ................................................................................... 1

Integrating RNAP II Transcription and Nuclear Processing Events .............. 6
Synthesis of Pre-mRNA ................................................................. 6
Transcription Elongation and Genome Maintenance ............................ 9
RNAP II Regulates Pre-mRNA Capping .......................................... 10
Pre-mRNA Splicing and Recruitment of the Spliceosome .................... 12
Regulation of Alternative Splicing by SR Proteins and hnRNPs .............. 15
ATP-dependent Remodeling of the Spliceosome during Splicing ............ 17
Terminal Exon Definition Requires the Cap and 3' Cleavage Site ........... 19
RNAP II and the Spliceosome Connect Splicing to Transcription ............ 21
3' End Cleavage and Polyadenylation ............................................ 23
Monitoring 3' End Integrity during Transcription Elongation .................. 27
Coordinating Nuclear Processing Events and mRNA Export ..................... 29
General Mechanisms of Nucear/Cytoplasmic Transport ...................... 29
mRNA Export Requires a Complex Array of Factors ........................... 31
Pre-mRNA Splicing Factors Are Required for mRNA Export ................. 33
TREX Links Transcription, Splicing and mRNA Export ........................ 37
Connections between Polyadenylation and mRNA Export ................... 38

MATERIALS AND METHODS .................................................................. 43

Yeast and Bacterial Culture Media ..................................................... 43
Yeast Strains and Plasmids ............................................................... 44
Nucleic Acid Isolation Procedures ....................................................... 47
Cell Transformation .......................................................................... 49


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Yeast Genetic Manipulations ............................................................. 50
Yeast Total Cell Protein Isolation ....................................................... 50
Fluorescence In Situ Hybridization and Cellular Immunofluorescence .......... 51
In Vitro 3' End Processing Assays ..................................................... 52
Filter Binding Assays ........................................................................ 53
Tandem Affinity Purification ................................................................. 53
Preparation of Polyclonal Antisera and Monoclonal Antibodies ................. 55
Poly(A) Tail Length Determination ..................................................... 56
In Vitro GST Pull-down Experiments ..................................................... 56
Phosphatase Treatment with X Phosphatase .......................................... 58

R E S U LT S ....................................................................................... .. 59

Research Objectives ........................................................................ 59
Nab2p Binds Poly(A) RNA and Limits Poly(A) Tail Length In Vitro .............. 61
Expression and Purification of Recombinant GST-Nab2p-His6 Protein ..... 62
rNab2p Binds with High Affinity to Poly(A) RNA Homopolymers ............. 62
Nab2p Restricts Poly(A) Tail Length In Vitro ..................................... 65
NAB2 Limits Poly(A) Tail Lengths and Promotes mRNA Export In Vivo ......... 72
Nuclear Targeting of Pabl p Suppresses the nab2A Growth Defect ......... 73
Nab2p Is Required For Poly(A) Tail Length Control and mRNA Export .... 76
Nab2p Associates with Factors Required for mRNA Export ...................... 85
Purification of Nab2pT-associated Proteins ....................................... 85
Nab2p and Kapl04p Form an Abundant Complex in Yeast Extracts ........ 89
Nab2p Co-purifies with Nuclear mRNA-binding Proteins ..................... 95
mRNA Export Is Inhibited by a Mutation in the NES of Nab2p ................ 100
Mex67p Interacts with the N-terminus of Nab2p .................................. 101
nab2-20 Stabilizes Mex67p-Nab2p Interactions In Vitro ......................... 104
Nab2p Is Phosphorylated in Strains Defective for mRNA Export ............. 110

D IS C U S S IO N ....................................................................................... 115

Nab2p Is Required for Poly(A) Tail Length Restriction ............................... 115
Potential Role for Nab2p in Preventing Nucleolar Retention of mRNA .......... 121 Nab2p Interacts with Import and Export Receptors ................................... 126
Lim itatio ns ....................................................................................... 130


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C o nclusio ns ..................................................................................... 132

A P P E N D IX .......................................................................................... 133

R E F E R E N C E S ..................................................................................... 138

BIOGRAPHICAL SKETCH ...................................................................... 167









































vi














Abstract of Dissertation Presented to the Graduate School
Of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
COORDINATION OF MRNA 3' END FORMATION AND NUCLEAR EXPORT BY
A NUCLEAR POLY(A)-BINDING PROTEIN

By

Keith Robert Nykamp

December 2003

Chair: Maurice S. Swanson
Major: Molecular Genetics and Microbiology

Eukaryotic messenger RNA is transcribed in the nucleus and translated into protein in the cytoplasm. Prior to export from the nucleus, mRNAs must be capped at the 5' end with a 7-methylguanylate (m7G), introns must be removed via splicing, and a polyadenylate tail must be added to the 3' end. Splicing and polyadenylation are carried out by large macromolecular machines and are coordinately regulated by RNA polymerase II (RNAP II) transcription. During transcription, members of a large family of nuclear RNA-binding proteins, the heterogeneous nuclear RNA binding proteins (hnRNPs), bind to nascent RNAP II transcripts. Co-transcriptional association of hnRNPs with nascent pre-mRNA is thought to regulate the specificity and timing of subsequent RNA processing events by influencing the recruitment of the spliceosome and cleavage/polyadenylation factors. Following pre-mRNA processing, export from


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the nucleus is promoted by interactions between the mRNA and specialized nuclear export factors.

Although hnRNPs associate with nuclear poly(A) RNA and are required for efficient mRNA export in vivo, their role in mRNA export has remained controversial. Suggestions have been made that hnRNPs bind non-specifically to pre-mRNA and do not directly recruit mRNA export receptors. Alternatively, hnRNPs may act at the interface between mRNA processing events and nuclear export, orchestrating the temporal recruitment of mRNA export factors after processing has occurred in vivo. The goal of the research presented in this report was to test the latter hypothesis using the yeast hnRNP Nab2p. My results demonstrate that Nab2p is a nuclear poly(A)-binding protein, required for both termination of polyadenylation and mRNA export. Surprisingly, these two processes can be uncoupled in nab2 mutant strains, and Nab2p interacts with the nuclear mRNA export factor Mex67p. Based on these results, the proposition is made that Nab2p coordinates the termination of polyadenylation with mRNA export in vivo.

















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INTRODUCTION

The synthesis of messenger RNA (mRNA) in a eukaryotic cell occurs in the nucleus and involves transcription of pre-mRNA by RNA polymerase II (RNAP II) followed by several processing steps, including capping, splicing, and polyadenylation (see Figure 1). The translation of mRNA into proteins, however, occurs in the cytoplasm. Importantly, nuclear pore complexes (NPCs) allow for the translocation of RNAs and proteins through the nuclear envelope (NE) (reviewed in Ryan and Wente, 2000).

Compartmentalization of transcription and pre-mRNA processing in the nucleus and protein translation in the cytoplasm allows for much greater regulation of gene expression than could be attained otherwise (reviewed in Hood and Silver, 1999; Komeili and O'Shea, 2000). For example, p53 normally shuttles between the nucleus and cytoplasm, but localizes to the nucleus during stress conditions where it increases the transcription of stress response genes (Middeler et al., 1997). Interestingly, the inability of p53 to localize in the nucleus correlates with the proliferation of several tumors emphasizing the importance of protein import regulation (Moll et al., 1995; Shlamp et al., 1997).

The addition of a 7-methylguanylate cap to the 5' end and a polyadenylate tail to the 3' end of mRNA provides additional opportunities for the eukaryotic cell to regulate gene expression (Shatkin and Manley, 2000). Under normal




1





2

conditions, the cytoplasmic cap-binding protein (elF4E) and poly(A) binding protein (PABP), protect against degradation and aid in protein synthesis through interactions with the cap and poly(A) tail, respectively (reviewed in Wilusz et al., 2001). AU-rich element (ARE) RNA-binding proteins, such as AUFI/hnRNP D and tristetraprolin (TTP) bypass PABP-dependent mRNA stabilization by promoting mRNA decay when bound to 3' untranslated (UTR) regions of protooncogene and cytokine mRNAs (Lai et al., 1999; Laroia et al., 1999; Loflin et al., 1999). Alternatively, HuR antagonizes the binding of AUF1/hnRNP D to AREs and augments PABP-dependent stabilization of these mRNAs (Fan and Steitz, 1998; Gallouzi et al., 2000). Other ARE-binding proteins (TIAR and TIA-1) have been demonstrated to inhibit PABP-dependent re-initiation of protein synthesis (Gueydan et al., 1999; Piecyk et al., 2000). Importantly, ARE mutations or aberrant levels of ARE-binding proteins correlate with a variety of diseases, including autoimmunity, arthritis, myeloid hyperplasia, and tumorigenesis (Gouble et al., 2002; Kontoyiannis et al., 1999; Taylor et al., 1996). Properly regulated mRNA turnover is clearly very important for cell viability.

Alternative pre-mRNA splicing also gives rise to extraordinary levels of gene regulation by increasing the number of different proteins that can be produced by a single gene (Smith and Valcarcel, 2000). The D. melanogaster Down's Syndrome cell adhesion molecule (Dscam) gene provides an extreme example of this phenomenon (Schmucker et al., 2000). The authors predict that

-38,000 distinct protein isoforms are generated by alternative pre-mRNA splicing. Given so much diversity and complexity, it is not surprising that





3


symptoms of many diseases have been correlated with splicing errors (reviewed in Faustino and Cooper, 2003). For example, mutations that eliminate or alter constitutive cis-acting splicing signals often result in abnormal mRNAs due to aberrant intron inclusion or exon exclusion. In most cases, these abnormal mRNAs are susceptible to degradation by nonsense-mediated decay (NMD) and result in lost function (Culbertson, 1999). If aberrantly spliced mRNAs are exported, however, they may generate truncated proteins with additional, possibly deleterious, functions.

While pre-mRNA processing events and nucleocytoplasmic transport provide many opportunities for gene regulation, they also create logistical problems for eukaryotic organisms. What mechanisms are in place to ensure that only mature, and not incompletely processed, mRNAs are exported to the cytoplasm? It turns out that proteins essential for transcription are also important for accurate pre-mRNA processing and factors vital for pre-mRNA processing events are necessary for efficient mRNA export (for reviews see Hirose and Manley, 2000; Jensen et al., 2003; Maniatis and Reed, 2002; Proudfoot et al., 2002; Steinmetz, 1997). Integrating transcription, pre-mRNA processing and mRNA export makes perfect sense because this linkage provides a mechanism for the eukaryotic cell to assess mRNA quality at each step of the gene expression pathway before proceeding to the next step.













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Inte-grating RNAP II Transcription and Nuclear Processing Events Synthesis of Pre-mRNA

RNAP II transcribes all eukaryotic pre-mRNA and is the primary target for gene regulation as well as the principal conductor of pre-mRNA processing events. Importantly, the mechanisms of pre-mRNA transcription are highly conserved among eukaryotes allowing researchers to study transcription initiation and elongation in a variety of model organisms. Genetic screens in D. melanogaster and S. cerevisiae, as well as biochemical strategies in human and yeast cells, have identified a holoenzyme consisting of five major protein complexes (RNAP II, General Transcription Factors (GTF), Srb-mediator; SWISNF, and CDK) required for transcription from chromatin-associated DNA templates (for reviews, see Conaway and Conaway, 1993; Kornberg et al., 1992; Lemon and Tjian, 2000; Myer and Young, 1998; Woychik and Hampsey, 2002). The core enzyme consists of twelve subunits all of which are required for enzymatic activity. In yeast, these subunits are encoded by genes RPB1 through RPB12. An interesting feature of RNAP II is a disordered COOH-terminal repeat domain (CTD) consisting of twenty-six consensus YSPTSPS heptad repeats encoded by the yeast RPB1 gene (Cramer et al., 2001). The CTD is unique to RNAP II and is central to the regulation of transcriptional initiation and elongation (Myer and Young, 1998; Price, 2000; Woychik and Hampsey, 2002).

Before transcription is initiated and synthesis of pre-mRNA begins, the holoenzyme assembles on the DNA at the site of transcription. RNAP II alone does not recognize specific DNA sequences and is unable to respond to





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transcriptional activators (Myer and Young, 1998). Promoter recognition requires the GTF complex (TFIID, TFIIB, TFIIF, TFIIH, and TFIIE) (Roeder, 1996; Woychik and Hampsey, 2002). DNA-binding activities associated with TFIID and TFIIB nucleate the formation of a pre-initiation complex (PlC) immediately upstream of the transcription start site. Addition of the remaining GTFs (TFIIF, TFIIE, and TFIIH) completes PIC assembly on the promoter. TFIIF plays a structural role in stabilizing PIC assembly while TFIIE and TFIIH eliminate transcription initiation obstacles, such as supercoiled DNA and DNA-bound nucleosomes, through ATP-dependent unwinding and remodeling activities (Dvir et al., 2001). SWI-SNF also utilizes ATP hydrolysis to facilitate initiation and elongation in the midst of chromatin barriers (Lemon and Tjian, 2000). Srbmediator receives signals from enhancer-bound activators and modulates PIC formation and transcription initiation through interactions with RNAP II and TFIIH (Woychik and Hampsey, 2002). Finally, the cyclin dependent kinase (CDK) and its cyclin partner regulate PIC assembly (Liao et al., 1995). Yeast cells lacking the CDK homologue (SrblOp) exhibit de-repression of highly repressed genes such as SPO013, GAL1, SUC2, PHO5, and MFA2 (Kuchin et al., 1995; Wahi and Johnson, 1995). SrblOp exerts its effect on transcription initiation by phosphorylating the CTD of RNAP II on Serine 5 (Ser5) of the heptad repeats (Hengartner et al., 1998). These findings, combined with the fact that RNAP II purified from PIC preparations does not contain phosphates (Kang and Dahmus, 1993), suggest a model in which SrblOp suppresses transcription from a subset of genes by blocking PIC formation. How is Srbl Op activity controlled to allow for





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transcription initiation of highly active genes? A phosphatase, Fcplp in yeast, interacts with TFIIF and has the ability to dephosphoyrlate the CTD at both Ser5 and Serine 2 (Ser2) of the heptad repeats (Cho et al., 2001). Fcplp may stimulate initiation by antagonizing the effects of SrblOp on Ser5. In addition, dephosphorylation of Ser2 by Fcpl may be required for recycling RNAP II for another round of transcription (see below).

After PIC formation, RNAP II escapes from the promoter and enters an early elongation phase. These steps require ATP-dependent helicase and CTD kinase activities associated with the transcription factor TFIIH (Dvir et al., 2001). In yeast, the product of the KIN28 gene co-purifies with TFIIH and kin28 mutations correlate with general transcription defects (Cismowski et al., 1995; Valay et al., 1995). Hengartner et al. (1998) have further demonstrated that Kin28p is responsible for phosphorylating Ser5 after PIC formation and may provide a signal for elongation to commence (Hengartner et al., 1998). Remarkably, the timing of Ser5 phosphorylation can have profound effects on pre-mRNA transcription. SrblOp mediated phosphorylation of Ser5 prior to PIC formation blocks transcription initiation while Ser5 phosphorylation by Kin28p after PIC formation stimulates elongation. Clearly, promoter escape and early elongation are highly regulated processes and these steps play an important role in regulating gene expression (Kugel and Goodrich, 1998; Kumar et al., 1998). For example, heat shock protein genes have stalled RNAP II complexes at their promoters with nascent (20-40 nucleotides) transcripts waiting to elongate in response to heat stress (Uptain et al., 1997). Defective elongation, as observed





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in Xeroderma Pigmentosum patients, gravely affects cell viability (Liu et al., 2001).

The phosphorylation status of Ser2 plays an important role in the transition from early to productive elongation. A Positive Transcription Elongation Factor b (P-TEFb) is required for the synthesis of long transcripts in mammals (Reines et al., 1999). P-TEFb is composed of a CDK subunit (Cdk9) and several cyclins and phosphorylates Ser2 of the CTD after initiation (Price, 2000) providing evidence that Ser2 phosphorylation is essential for efficient elongation. In yeast, the Cdk9 homolog (Ctkl p) is not essential for vegetative growth and presumably not required for transcription elongation (Sterner et al., 1995). Corden and colleagues have found that Ctklp regulates transcription during heat shock and under nutritional stress (Patturajan et al., 1999; Patturajan et al., 1998). The authors propose that Ctkl p may be recruited to specific promoters in conjunction with RNAP I1. Transcription elongation of these genes is then regulated by Ctklp-dependent phosphorylation of Ser2. Importantly, these results provide evidence that transcription is regulated not only before initiation begins but also during elongation.

Transcription Elonqation and Genome Maintenance

Productive elongation is a very complex process and other factors have been identified that facilitate elongation by removing protein obstacles from the DNA, alleviating torsional strain caused by DNA supercoiling, or by interacting with RNAP II to suppress transcriptional pausing and arrest (Reines et al., 1999). SWI-SNF is essential for the formation of the PIC, promoter clearance and





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elongation of certain genes (Wolffe, 1994). FACT was purified by virtue of its ability to facilitate transcriptional elongation from chromatin templates in vitro using only RNAP II and GTFs (Orphanides et al., 1998). SII (PPR2 in yeast) directly interacts with RNAP II to activate cleavage of stalled transcript so that the enzyme can re-extend, allowing repeated attempts at elongation. In yeast, the THO complex also plays a role in elongating through transcriptional pauses (Chavez et al., 2000). Mutations in members of the THO complex, such as Hprl p and Tho2p, result in defective elongation of a lacZ reporter gene, although elongation through several other yeast genes is unaltered (Piruat and Aguilera, 1998). Mutant hprl and tho2 strains are also hypersensitive to the elongation inhibitor, 6-azauracil, indicating THO facilitates productive elongation through transcriptional blocks. Interestingly, hprl mutants also exhibit hyperrecombination phenotypes (Chavez and Aguilera, 1997). The authors believe that when transcription is hindered due to elongation impediments, such as long DNA repeats, the transcribed region becomes highly susceptible to recombination. Indeed, a stalled RNAP II may provide a signal to the DNA repair machinery that a region of the genome is unstable and needs to be repaired by homologous recombination.

RNAP II Regulates Pre-mRNA Capping

The addition of a 7-methylguanylate (m7G) to the 5' end of a pre-mRNA is the first step in the production of mature mRNA (for reviews see Shatkin, 1976; Shuman, 2001). Cap addition is a three-step process carried out by three distinct enzymes in yeast. An RNA triphosphatase (Cetl p) hydrolyzes the y-phosphate





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from the 5' end of the pre-mRNA and a guanyltransferase (Cegip) links a GMP to the resulting diphosphate end via a 5' to 5' phospoanhydride bond (Shibagaki et al., 1992; Tsukamoto et al., 1997). A methyltransferase (Abdlp) rounds out the capping process by transferring a methyl group to the guanine on position 7 (Mao et al., 1995).

It has been known for a long time that capping occurs co-transcriptionally when nascent RNA chains are between 25 and 30 nucleotides in length (Coppola et al., 1983; Jove and Manley, 1984). How is the mRNA capping process coordinately regulated with transcription? Several studies have provided evidence that all three capping enzymes selectively associate with RNAP II only when the heptad repeats are phosphorylated at the Ser5 position (Komarnitsky et al., 2000; McCracken et al., 1997; Rodriguez et al., 2000; Shroeder et al., 2000). Further, Ceglp activity is dependent on the presence of Cetlp and increases with higher levels of phosphorylation at Ser5, but not Ser2 (Cho et al., 2001; Ho et al., 1998; Ho and Shuman, 1999). Thus, the capping apparatus is recruited by RNAP II to the site of transcription and allosterically activated before transcription elongation commences.

The benefits of coupling transcription and capping are not biased toward the capping reaction. Interactions among Cetlp, Ceglp and the CTD also serve as a checkpoint for productive elongation. Myers et al. (2002) identified the Cetlp-Ceglp heterodimer as a repressor of transcription elongation and reinitiation in vitro (Myers et al., 2002). Apparently, the transcription machinery is unable to carry on with transcription if Abdlp is not present to methylate the





12

guanylate cap. In support of these results, Jove and Manley demonstrated twenty years ago that inhibiting transmethylation by adding Sadenosylhomocysteine to transcription reactions in vitro suppressed initiation (Jove and Manley, 1982).

Proper cap formation is essential for cellular viability and has been demonstrated to be important for mRNA stability and turnover, pre-mRNA processing, and translation initiation (Gallie, 1998; Lewis and Izaurralde, 1997; Shatkin, 1985; Shatkin and Manley, 2000; Tucker and Parker, 2000; Wilusz et al., 2001). These cellular functions can be attributed to proteins that specifically recognize the m7G cap in the nucleus and the cytoplasm (Marcotrigiano et al., 1997; Mazza et al., 2002). In the nucleus, a heterodimeric complex of 80 (CBP80) and 20 (CBP20) kDa subunits binds the cap and stimulates splicing and polyadenylation (Cooke and Alwine, 1996; Izaurralde et al., 1994) (also see below). In the cytoplasm, the cap is bound by a single 25 kDa polypeptide (elF4E) that stimulates protein translation through its interactions with the cap, PABP and several translation initiation factors (Sonenberg, 1996). Pre-mRNA Splicinq and Recruitment of the Spliceosome

In an astonishing discovery twenty-six years ago, mRNA sequences (exons) of eukaryotic genes were found to be interrupted throughout the DNA by non-coding segments (introns) (Berget et al., 1977; Chow et al., 1977). Exons are joined together in a contiguous manner via two sequential transesterification reactions (Burge et al., 1999; Kramer, 1996; Sharp, 1994). In the first reaction, the 2' hydroxyl of a conserved branchpoint adenosine attacks the phosphate





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group of the 5' splice-acceptor site. This results in two molecules: (1) exon 1

(El) with a free 3' hydroxyl and (2) a lariat intermediate formed by a unique 5' to 2' linkage between the first nucleotide of the intron and the branchpoint adenosine. In the second reaction, the 3' hydroxyl of El attacks the 3' splicedonor site of exon 2 (E2) generating a ligated mRNA and a free intronic lariat. The first transesterification reaction has been shown to occur in vitro in the absence of proteins and ATP (Valadkhan and Manley, 2001; Valadkhan and Manley, 2003) providing evidence that catalysis is RNA-mediated. However, five small nuclear RNAs (Ul, U2, U4, U5, U6) and approximately 100 proteins assemble into a large, energy consuming, splicing machine (spliceosome) on the pre-mRNA and increase the efficiency and fidelity of the transesterification reactions both in vitro and in vivo (for reviews see Adams et al., 1996; Burge et al., 1999; Hastings and Krainer, 2001; Kramer, 1996; Madhani and Guthrie, 1994; Staley and Guthrie, 1998; Will and Luhrmann, 2001).

Spliceosome assembly begins with the selection of a 5' splice site and the formation of a commitment complex (CC) (Michaud and Reed, 1991; Seraphin and Rosbash, 1989). Commitment complex formation specifies the boundaries between introns and exons and is possibly the most important step in the regulation of splicing. As mentioned earlier, inappropriate splice site selection can be very detrimental to the health of a eukaryotic cell. How are relatively short splicing signals recognized within a sea of RNA? In yeast, CC assembly is initiated by the formation of a duplex between UI snRNA and a conserved GUAUGU sequence adjacent to the 5' splice site (Zhang and Rosbash, 1999).





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Concurrent with U1 binding, a branchpoint binding protein (BBP) binds the branchpoint adenosine sequence (UACUAAC) (Berglund et al., 1997) and Mud2p binds a polypyrimidine tract located between the branchpoint and 3' splice site (AG dinucleotide) (Abovich et al., 1994). Importantly, interactions among the U1 snRNP, BBP, and Mud2p define introns within the pre-mRNA and provide a scaffold for the recruitment of U2 snRNP (Abovich and Rosbash, 1997) (also see below).

In metazoan organisms, highly degenerate splice site and branchpoint sequences amid very long introns (> 1000 nucleotides) make intron recognition much more difficult (Berget, 1995; Reed, 1996). Berget and colleagues proposed that vertebrate splice site recognition requires interactions between the 3' splice site of the upstream intron and the 5' splice site of the downstream intron for the definition of exons (Robberson et al., 1990). In support of this model, SR proteins, characterized by RNA recognition motifs (RRM) and arginine/serine-rich (RS) domains, bind exonic splicing enhancers (ESEs) and participate in CC formation (Fu, 1995; Graveley, 2000). Both SF2/ASF and SC35 interact with the U1-70K U1 snRNP protein through RS domains (Jamison et al., 1995; Kohtz et al., 1994; Staknis and Reed, 1994) suggesting these proteins facilitate the early recognition of 5' splice sites. Furthermore, U1 depletion and U1 snRNA antisense experiments have demonstrated that SR proteins, in particular SC35, promote CC formation even in the absence of functional U1 snRNPs (Crispino et al., 1993; Tarn and Steitz, 1994).





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Recognition of the mammalian branchpoint region also requires branchpoint and pyrimidine tract binding proteins, mBBP and U2 auxilliary factor (U2AF), respectively (Abovich et al., 1994; Arning et al., 1996; Berglund et al., 1997; Ruskin et al., 1988). U2AF is composed of two subunits, U2AF65 and U2AF35 (Zamore and Green, 1989). U2AF65 is most similar to yeast Mud2p and binds directly to the polypyrimidine tract and 3' splice site region of the intron (Abovich et al., 1994; Singh et al., 1995). U2AF35 is unique to metazoan organisms and physically interacts with both SC35 and SF2/ASF (Fu and Maniatis, 1992; Wu and Maniatis, 1993) providing an explanation for the observation that both 5' and 3' splice sites are required for CC formation in mammals (Michaud and Reed, 1993; Reed, 1996). Thus, SR proteins bind to sequences within mammalian exons and bridge 3' and 5' splice sites thereby facilitating exon recognition.

Regulation of Alternative Splicing by SR Proteins and hnRNPs

Given their function in 5' splice site recognition and exon definition, it is not surprising that SR proteins also influence the selection of alternative splice sites (Black, 2003; Faustino and Cooper, 2003; Grabowski and Black, 2001; Smith and Valcarcel, 2000). In addition, heterogeneous nuclear RNA binding proteins (hnRNPs) associate with the pre-mRNA during transcription and, in several cases, antagonize the binding of SR proteins to the pre-mRNA (Dreyfuss et al., 2002; Dreyfuss et al., 1993; Krecic and Swanson, 1999). When one of two identical 5' splice sites needs to be paired with a single 3' splice site in vitro, high concentrations of hnRNP Al in splicing extracts inhibits the binding of Ul snRNP





16


to both 5' splice sites (Eperon et al., 2000). However, an ESE placed near one 5' splice site results in the recruitment of U1 snRNP to that site and precipitates the formation of a CC on the pre-mRNA (Eperon et al., 2000). Importantly, ESE effects are dependent on the addition of recombinant SF2/ASF to extracts. These findings have led to the proposal that hnRNP Al, and possibly all hnRNPs, bind indiscriminately to pre-mRNA, block binding of sequence-specific RNA-binding proteins (i.e. U1 snRNP and SR proteins) and inhibit the inclusion of alternatively spliced exons (Hastings and Krainer, 2001). In support of this model, SF2/ASF displaces hnRNP Al from ESEs when present in extracts at high concentrations (Eperon et al., 2000; Zhu et al., 2001). Although this model adequately explains the experimental observations of the Krainer group, it is not in accordance with findings reported by Black and colleagues. For example, Rook et al. (2003) described a purine-rich ESE required for inclusion of the neuronal-specific exon (Ni) of the c-src gene (Rooke et al., 2003). UVcrosslinking and immunoprecipitation experiments have indicated that SF2/ASF, hnRNP Al, H and F all interact with the NI ESE. Consistent with previous studies, SF2/ASF stimulates splicing and hnRNP Al represses the inclusion of Ni. However, hnRNP H and F are both components of the enhancer complex and required for N1 inclusion (Chou et al., 1999). In addition, a C to T mutation in exon 7 of the SMN2 gene converts the surrounding sequence into an hnRNP Al binding site leading to exclusion of exon 7 from SMN2 mRNA (Kashima and Manley, 2003). Taken together, these studies provide evidence that hnRNPs are not to be dismissed as generic RNA-binding proteins and general repressors of





17


sequence-specific RNA-binding proteins. Rather, they bind defined sequences and perform specialized roles in the regulation of alternative splicing.

It is important to point out that very few yeast genes contain introns. Of the genes that do yield spliced mRNAs, most have consensus 5' splice-sites, very few have multiple introns and only one or two genes encode alternatively spliced mRNAs (Davis et al., 2000). This may explain why only a few hnRNP and SR proteins have been discovered in yeast and suggests that these proteins will play important roles in other pre-mRNA processing events, besides splicing (Anderson et al., 1993b; Wilson et al., 1994; Windgassen and Krebber, 2003; Zenklusen et al., 2001).

ATP-dependent Remodeling of the Spliceosome during Splicinq

After 5' splice site recognition, the pre-spliceosome is formed by a duplex between the U2 snRNA and branchpoint sequence (Kramer, 1996; Staley and Guthrie, 1998). Before the U2 snRNP can bind to the branchpoint, however, two energy-dependent, structural rearrangements must occur. First, the branchpoint base-pairing sequence of U2 snRNA must be prepared for binding. Second, Mud2p/U2AF65 and BBP need to be removed from the U2 snRNA pre-mRNA binding site. In yeast, Prp5p modulates the former rearrangement and the latter is coordinated by Sub2p (Kistler and Guthrie, 2001; Libri et al., 2001; O'Day et al., 1996; Zhang and Green, 2001). Both of these proteins are members of the DExD/H-box RNA helicase superfamily characterized by conserved ATPase, helicase and RNA interaction motifs (de la Cruz et al., 1999; Tanner and Linder, 2001). Temperature-sensitive mutations in prp5 result in pre-spliceosome





18

formation defects in vitro and are lethal in combination with mutant U2 alleles (Ruby et al., 1993; Wells and Ares Jr., 1994; Yan and Ares Jr., 1996). Subsequent experiments have demonstrated that Prp5p is responsible for an ATP-dependent structural change in the intact U2 snRNP prior to branchpoint binding (O'Day et al., 1996). Furthermore, mutations that lead to misfolding of U2 snRNA are suppressed by gain-of-function mutations in cus2 while loss-offunction alleles of cus2 are lethal in combination with both U2 and prp5 mutations (Perriman and Ares Jr., 2000; Yan et al., 1998). Remarkably, pre-spliceosome assembly does not require ATP in cus2A extracts indicating that Cus2p is responsible for rendering pre-spliceosome formation dependent on ATP (Perriman and Ares Jr., 2000; Yan et al., 1998). The authors of these studies propose that Cus2p binds U2 snRNA and inhibits branchpoint binding until Prp5p removes Cus2p through ATP-dependent remodeling activities. SUB2 is homologous to the human gene UAP56 encoding the U2AF65-interacting protein and genetically interacts with MUD2 (Fleckner et al., 1997; Kistler and Guthrie, 2001). Although SUB2 is essential for cell viability, deletion of MUD2 allows for growth in the absence of SUB2. Cus2p and Mud2p appear to provide ATPdependent checkpoints during the splicing process allowing splicing to be halted, if necessary, through regulatory control of Prp5p and Sub2p, respectively.

Four more ATP-dependent remodeling and assembly steps are required for the transesterification steps to proceed. The U1 snRNA interaction with the 5' splice-site must be exchanged for a mutually exclusive interaction with the U6 snRNA (Kandels-Lewis and Seraphin, 1993; Konforti et al., 1993). The U6





19


snRNA is not recruited to the spliceosome alone. Rather, the U4/U6oU5 trisnRNP escorts U6 to the pre-spliceosome (Kramer, 1996). For the spliceosome to become catalytically active, an extremely stable base-pair interaction between U4 and U6 snRNAs must be disrupted (Nilsen, 1998). After activation of the spliceosome, both transesterification reactions require extensive remodeling of the pre-mRNA in order to proceed in vivo (Staley and Guthrie, 1998). Prp28 removes Ul from the 5' splice site (Staley and Guthrie, 1999), Brr2 unwinds the U4/U6 RNA duplex (Raghunathan and Guthrie, 1998), and Prp2 restructures the pre-mRNA during the first transesterification reaction while Prp16 does so during the second reaction (Kim and Lin, 1996; Schwer and Guthrie, 1991). Terminal Exon Definition Requires the Cap and 3' Cleavage Site

As discussed above, accurate splicing requires the efficient assembly of a commitment complex on the pre-mRNA that distinguishes exons from introns. According to the exon definition model, proteins bind to introns near the 5' and 3' splice sites and interact with ESE bound proteins to form a network of interactions across the exon. For internal exon definition, the supporting cast of characters include U1 snRNP, U2AF, SR proteins and hnRNPs. Two additional exons exist in the pre-mRNA and require a different collection of proteins for their definition. A 5' terminal exon is defined by the 5' cap and 5' splice site within the first intron and a 3' terminal exon is demarcated by a 3' splice site of the last intron and a 3' end cleavage site. In order for the exon definition model to be correct, cap-binding proteins and 3' end formation factors must also be required for splicing fidelity and efficiency. In fact, an interaction between the human





20


CBC and hnRNP F has been described (Gamberi et al., 1997). HnRNP F preferentially associates with CBC-RNA complexes rather than naked RNA and depletion of hnRNP F from HeLa cell nuclear extracts reduces splicing efficiency in vitro. How does the CBC-hnRNP F interaction influence splicing activity? Black and colleagues have demonstrated that hnRNP F forms a complex with hnRNP H and SF2/ASF on the N1 exon of the c-src pre-mRNA to promote exon inclusion (Chou et al., 1999). Possibly, when bound to ESEs, hnRNP F and SF2/ASF form a network of interactions to define the 5' terminal exon: hnRNP F interacts with CBC and SF2/ASF recruits the Ul snRNP to the 5' proximal splice site. Studies in yeast also indicate that connections between the CBC and U1 snRNP are necessary for delineating the cap-proximal exon. Using in vivo reporter assays, Fortes et al. (1999) demonstrated that LUC7 is required for nonconsensus splice site recognition and luc7 mutant strains fail to utilize capproximal splice sites if two competing 5' splice sites are present (Fortes et al., 1999a). Luc7p is a U1 snRNP-associated protein with an SR-like domain and is indispensable for CC formation in vitro as well as Ul snRNP-CBC interactions in vivo (Fortes et al., 1999a). In a second study by the same group, cell growth was compromised in a mud2A strain if both cbp80 and cbp20 were also deleted from the genome (Fortes et al., 1999b). Importantly, growth is only mildly affected in either mud2A or cbp8OAcbp2OA strains.

For 3' terminal exon definition, an interaction between U2AF65 and poly(A) polymerase (PAP) in HeLa cells has been described (Vagner et al., 2000). PAP stimulates the binding of U2AF65 to the pyrimidine tract of the





21

terminal intron. These interactions appear to be specific for metazoan organisms because no interactions between Mud2p and yeast PAP have been described. Furthermore, Dower and Rosbash (2002) observed efficient splicing in vivo in the absence of both pre-mRNA capping and polyadenylation. Since yeast introns are small, splicing signals are highly conserved, and most introns are near the 5' end of the gene (Davis et al., 2000), interactions between the spliceosome and 3' end formation complex may not be essential for efficient splicing in yeast. In fact CBC-spliceosome interactions only appear to be essential when non-consensus signals are utilized lending credence to the idea that exon definition is critical for recognition of weak, alternatively used splice sites, such as those primarily found in metazoan organisms.

RNAP II and the Spliceosome Connect Splicingq to Transcription

Considering synthesis of pre-mRNA is carried out solely by RNAP II, it is not surprising that RNAP II has been reported to interact with components of the spliceosome. For example, reporter genes containing introns are not spliced in metazoan cells if transcribed by RNAP I or RNAP III (Sisodia et al., 1987; Smale and Tjian, 1985). Early in situ studies using Chironimus tentans Balbiani ring genes to visualize pre-mRNA processing events during transcription discovered that genes were spliced concurrently with transcription (Bauren and Wieslander, 1994; Kiseleva et al., 1994). In addition, phosporylated RNAP II (RNAP 110) increases the efficiency of splicing both in vivo and in vitro (Fong and Bentley, 2001; Fong et al., 2003; McCracken et al., 1997; Misteli and Spector, 1999). Alternatively, the addition of hypophosphorylated RNAP II (RNAP IIA) inhibits





22

splicing in vitro (Hirose et al., 1999; Zeng and Berget, 2000). In yeast, Prp40p is recruited to RNAP IIO, but not RNAP IIA (Morris and Greenleaf, 2000). Prp40p is a component of the U1 snRNP and directly interacts with BBP (Abovich and Rosbash, 1997). During transcription elongation, the U1 snRNP-Prp40p complex is tethered to RNAP II increasing the likelihood of 5' splice site recognition. Interactions between RNAP IIO and U1 snRNP also serve to retain the 5' splice site near elongating RNAP II, allowing branchpoint and 3' splice site sequences to be recognized by BBP and Mud2p immediately after synthesis. After binding to the pre-mRNA, U1 snRNP, Mud2p and BBP precipitate the formation of a commitment complex.

Interactions between RNAP II and the spliceosome also influence transcription re-initiation and elongation. TFIIH interacts with U1 snRNA and promoter-proximal 5' splice sites stimulate transcription re-initiation in a Uldependent manner (Kwek et al., 2002). Two factors required for efficient elongation, SII and TAT-SF1, also co-purify with U2 snRNP and RNAP IIO in vivo (Fong and Zhou, 2001; Robert et al., 2002). TAT-SF1 stimulates transcription elongation in vitro through interactions with P-TEFb (Price, 2000). In yeast, the TAT-SF1 homolog (CUS2) plays a mechanistic role in pre-spliceosome formation (Yan et al., 1998). Altogether, these findings provide for an appealing model in which Cus2p/TAT-SF1 and U2 snRNP are recruited to RNAP IIO and stimulate transcription elongation most likely through interactions with Ctklp/P-TEFb. Importantly, Cus2p/TAT-SF1 may inhibit association of U2 snRNP with the premRNA until BBP and Mud2p/U2A65 bind to the branchpoint region of the intron.





23

Assuming commitment complex formation occurs without difficulty, Prp5p/hPRP5 remodels the U2 snRNP by removing Cus2p/TAT-SF1 and Sub2p/UAP56 dissociates Mud2p/U2A65 from the branchpoint allowing for assembly of the prespliceosome.

3' End Cleavaqe and Polyadenylation

Termination of RNAP II transcription coincides with cleavage of the premRNA at sequences downstream of the translation stop codon. Cleavage is followed by the addition of a polyadenylate tail of 70-90 nucleotides in yeast and 200-300 nucleotides in mammals (Proudfoot et al., 2002; Zhao et al., 1999). Like splicing, large protein complexes execute the cleavage and polyadenylation reactions with precision (Colgan and Manley, 1997; Wahle and Keller, 1996). Much has been learned about the factors responsible for cleavage and polyadenylation because many of these proteins are conserved from yeast to humans (see Figure 2) (Keller and Minvielle-Sebastia, 1997; Manley and Takagaki, 1996). In yeast, an A/U-rich Efficiency Element (EE) and an A-rich Positioning Element (PE) found upstream of the cleavage site recruit Cleavage Factor IB (CF IB) to the pre-mRNA. A second factor, Cleavage and Polyadenylation Factor I (CPF) associates with sequences surrounding the Pyrimidine-Adenosine (PyA) cleavage site (Proudfoot and O'Sullivan, 2002; Wahle and Ruegsegger, 1999). Interestingly CF IB is a single subunit factor composed of the yeast hnRNP Nab4p/Hrplp (Kessler et al., 1997). Nab4p controls cleavage site selection in vitro and in vivo and increases the efficiency of polyadenylation in vitro by interacting with the EE and recruiting CF IA and CPF











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26

to the mRNA (Chen and Hyman, 1998; Gross and Moore, 2001; MinvielleSebastia et al., 1998; Valentini et al., 1999). Thus, analogous to their role in selection of alternative splice sites, hnRNPs also control the fidelity of 3' end cleavage site selection. After CF IB, CF IA and CPF bind to the pre-mRNA, cleavage is carried out by an unknown endonuclease (CF II) providing a free 3' end for the addition of adenosines by PapIlp (Wahle and Ruegsegger, 1999).

Poly(A) addition in mammalian cells is initially a slow process with PAP dissociating from the growing tail after the addition of each nucleotide. After 10 or more adenylate residues have been added, however, a nuclear poly(A)binding protein (PABPN1, formerly PAB II) interacts with the nascent poly(A) tail, the cleavage and polyadenylation specificity factor (CPSF), and PAP to form a quaternary structure which transforms PAP into a processive enzyme (Bienroth et al., 1993; Murthy and Manley, 1995; Wahle, 1991). After approximately 200 nucleotides, the interaction between PABPN1 and CPSF is disrupted, thus terminating the polyadenylation reaction (Wahle, 1995). What is the mechanism of CPSF-PABPN1-PAP complex disruption? Using electron microscopy (EM), an oligomeric PABPN1 particle has been observed to co-localize with the growing poly(A) tail (Keller et al., 2000). Attractively, the maximum diameter observed for a PABPN1 particle (-21 nm) correlates with the particle size of a PABPN1poly(A) complex when the poly(A) molecule is 200-300 nucleotides in length. Longer poly(A) molecules do not result in larger PABPN1 particles suggesting the maximum PABPN1 particle size restricts poly(A) tail lengths to 300 nucleotides in vivo. These authors propose a model whereby PABPN1 initially





27

converts PAP into a processive enzyme via interactions with CPSF, but these interactions are not feasible when the PABPN1 particle reaches a maximum size, thus converting PAP back into a distributive enzyme.

In yeast, an essential poly(A)-binding protein (Pabip) has also been described, although Pablp shares minimal sequence similarity with PABPN1 (Sachs et al., 1986; Wahle, 1991). Pablp interacts in the yeast two-hybrid system with the CF IA component Rnal5p (Amrani et al., 1997) and recombinant Pablp limits poly(A) tail lengths in vitro (Minvielle-Sebastia et al., 1997). The role of Pablp in poly(A) tail length control in vivo, however, is questionable because the steady state distribution of Pablp is cytoplasmic (Anderson et al., 1993a). Furthermore, the examination of poly(A) tail lengths of total RNA in pablA cells reveals modest increases (-40 nucleotides) in vivo, whereas precursor RNAs have poly(A) tails greater than 200 nucleotides in pablA extracts (MinvielleSebastia et al., 1997; Sachs and Davis, 1989). Together, these results suggest that poly(A) tail length control in vivo is likely to require additional factors. Monitoring 3' End Integrity during Transcription Elongation

How are transcription and 3' end processing events coupled in vivo? The same general theme holds true for 3' end processing as described previously for capping and splicing. The CTD of mammalian RNAP II is essential for efficient cleavage at poly(A) sites and yeast strains containing CTD truncation mutants exhibit reduced cleavage efficiency (Hirose and Manley, 1998; Licatalosi et al., 2002; McCracken et al., 1997; Ryan et al., 2002). Components of the cleavage and polyadenylation machinery in yeast, including Rnal5p of CF IA and





28

Yhhlp/Cftlp of CPF, co-purify with RNAP IIO in vivo and directly interact with the phosphorylated CTD in vitro (Barilla et al., 2001; Dichtl et al., 2002b).

A conserved network of interactions between several GTFs and factors involved in cleavage and polyadenylation also exists to link transcription to 3' end formation. Dantonel et al. (1997) immunopurified CPSF with TFIID from HeLa cells. After transcription initiates, however, CPSF dissociates from TFIID and remains associated with RNAP IIO. In addition, PC4, a mammalian transcription co-activator, binds double-stranded DNA, interacts with TFIIA and RNAP IIO, and is phosphorylated by TFIIH in vitro (Malik et al., 1998; Werten et al., 1999). PC4 also associates with the 64 kDa subunit of CstF suggesting that CstF is recruited to the PIC as well (Calvo and Manley, 2001). These interactions have been conserved throughout evolution because the yeast homologue of PC4 (Sublp) binds to the CstF-64 counterpart (Rnal5p) in vitro (Calvo and Manley, 2001). SUB1 and SSU72 interact with TFIIB to assist in PIC assembly (Sun and Hampsey, 1996). Interestingly, mutations in these genes disrupt the fidelity of start site selection (Wu et al., 1999). Ssu72p also associates with two subunits of CPF (Ptalp and Ydhlp/Cft2p) and is required for CPF-dependent cleavage of the 3' end (Dichtl et al., 2002a; He et al., 2003). Together, Ssu72p and Sublp provide transcription start site specificity for RNAP II and recruit CF IA and CPF to the site of transcription. In addition, the presence of CPF at the site of transcription may provide a signal for RNAP II to begin elongation because Ptalp also interacts with Kin28p (Rodriguez et al., 2000). Phosphorylation of the CTD at Ser5 by Kin28p triggers the commencement of transcription elongation





29

(Hengartner et al., 1998). In the absence of efficient cleavage and polyadenylation by CPF, Kin28p may not be able to phosphorylate RNAP II, thereby halting transcription elongation. Thus, Ssu72p and Sublp ensure that newly synthesized mRNAs are correctly cleaved and polyadenylated

Coordinating Nuclear Processinq Events and mRNA Export General Mechanisms of Nuclear/Cytoplasmic Transport

After the 3' end has been cleaved and a poly(A) tail has been added the mature mRNA is ready for export. How are molecules transported into and out of the nucleus? The current model for general nuclear transport proposes that an adapter binds both cargo and a receptor. Several adapters and receptors have been identified in a number of different organisms that also associate with integral NPC proteins, or nucleoporins (Nups) (reviewed in Gorlich and Kutay, 1999; Komeili and O'Shea, 2001; Nakielny and Dreyfuss, 1999; Ohno et al., 1998; Pemberton et al., 1998). In addition to adapters and receptors, protein transport requires the GTPase Ran (Kuersten et al., 2001). Typically for protein import, an adapter (importin a) binds to a nuclear localization sequence (NLS) exemplified by the single SV40 large T antigen (PKKKRKV) or bipartite nucleoplasmin (KRPAAIKKAGQAKKKK) sequences and forms a quaternary structure with an import receptor (importin P), the NLS-containing protein, and RanGDP (Gorlich and Mattaj, 1996; Weis, 1998). A cytoplasmic GTPase Activating Protein (RanGAP) facilitates hydrolysis of GTP by Ran ensuring that RanGDP molecules accumulate in the cytoplasm (reviewed in Azuma and Dasso, 2000; Kuersten et al., 2001). Removal of GDP from Ran, in turn, requires





30

a nuclear guanine exchange factor (RanGEF) resulting in the accumulation of RanGTP in the nucleus. Assembly of an import complex is strengthened in the cytoplasm by RanGDP and recruited to the NPC by interactions between importin 13 and nucleoporins. After translocation through the nuclear pore, exchange of GDP for GTP in the nucleus releases the imported NLS protein from importin a, importin 13, and Ran (Ohno et al., 1998). Although several importin a and importin P3 proteins have been identified in metazoan organisms, the yeast genome encodes one importin a (Srpl) and one importin 13 (Kap95) (Gorlich and Kutay, 1999). Kap95p physically interacts with Nup116p and co-localizes with Srplp near the NPC in vivo (lovine et al., 1995; Koepp et al., 1996). Proper localization of Kap95p, Srplp, and NLS-containing proteins require a functional GTPase activating protein (Rnalp) and a functional guanine exchange factor (Prp20p) (Koepp et al., 1996).

Similar principles apply for export of proteins from the nucleus. Coprecipitation experiments first identified a human nuclear factor, homologous to importin 13, associated with NUP214/CAN and NUP88 (Fornerod et al., 1997b). The human gene was named hCRM1 because, in addition to having similarity to importin 13, the open reading frame was homologous to a previously identified gene in Schizosaccharomyces pombe (CRM1+). Early studies of CRMI+ indicated that it encoded a nuclear protein required for efficient DNA, RNA, and protein synthesis and condensation of chromosomal regions was observed in CRM1+ mutants at non-permissive temperatures (Adachi and Yanagida, 1989). Increased expression of CRM1+ was subsequently found to confer resistance to





31

leptomycin B (LMB), an agent known to induce cell cycle arrest at G1 and G2 phases of the cell cycle (Nishi et al., 1994). Analogous to protein import, a leucine-rich nuclear export sequence (NES) was discovered in the HIV-1 Rev and kinase A inhibitor PKI proteins (Fischer et al., 1995; Wen et al., 1995). In Xenopus oocytes and permeabilized HeLa cells, CRM1 has been shown to mediate the export of reporter proteins containing an NES while LMB strongly inhibited export of NES peptides (Fornerod et al., 1997a; Fukada et al., 1997; Ossareh-Nazzari et al., 1997). CRM1 exhibited cooperative interactions in vitro with both NES peptides and RanGTP providing evidence that CRM1 behaves like a traditional transport receptor (Fornerod et al., 1997a). mRNA Export Requires a Complex Array of Factors

In the same study, Fornerod et al. (1997) demonstrated that U snRNA export, but not mRNA export, was stimulated in Xenopus oocytes by increased levels of CRM1 and inhibited by LMB. In metazoan organisms U snRNAs are exported to the cytoplasm after transcription, assembled into functional snRNPs, and imported into the nucleus (reviewed in Paushkin et al., 2002; Will and Luhrmann, 2001). Four of the five spliceosomal U snRNAs (U1, U2, U4, and U5) are transcribed by RNAP II and have a m7G-cap to which the nuclear CBC is able to bind. In addition to CRM1, the CBC also plays an important role in the export U snRNAs (Hamm and Mattaj, 1990; Jarmolowski et al., 1994). In an elegant study, the Mattaj group provided the final piece in the puzzle of U snRNA export by identifying an adaptor protein (PHAX) which has the ability to bridge CRM1 and CBC interactions (Ohno et al., 2000). PHAX contains a leucine-rich





32

NES and formation of an snRNA-CBC-PHAX-CRM1 export complex required RanGTP. Remarkably, PHAX was unable to bind to CRM1, even in the presence of U snRNP, CBC, and RanGTP proteins, if it was not phosphorylated. These results provided evidence for an additional level of regulation for U snRNP export.

At the same time CRM1 was discovered in metazoans, Stade et al. (1997) reported that Crmlp was responsible for the export of NES peptides in yeast. Intriguingly, these authors also observed a block to poly(A) RNA export in a conditional crml mutant strain suggesting that CRM1 may serve as a receptor for mRNA export in yeast. A subsequent study by Neville and Rosbash (1999), however, demonstrated that a strain with an LMB-sensitive Crmlp continued to synthesize proteins in the presence of LMB arguing against a direct role for Crmlp in mRNA export. The discrepancy between these two reports can be explained by the finding that increased expression of a cytoplasmic DExD/H-box helicase (DBP5), required for proper poly(A) RNA localization, suppresses the crml-dependent mRNA export block (Hodge et al., 1999). Thus, mutations in CRM1 may indirectly affect mRNA export by altering the sub-cellular localization of cytoplasmic mRNA export factors.

What are the receptors and adapters for mRNA export? A synthetic lethal screen using a nup85 mutant allele identified MEX67 as a gene required for mRNA export in yeast (Segref et al., 1997). Mex67p interacts with Mtr2p to form a heterodimer localized to the nuclear periphery in vivo (Santos-Rosa et al., 1998). In addition to Nup85p, Mex67p-Mtr2p physically associates with several





33

other nucleoporins in vitro and mutations in mtr2 profoundly affect the localization of both Mex67p and poly(A) RNA in vivo (Santos-Rosa et al., 1998; Strasser et al., 2000). A human protein (TAP), with similarity to Mex67p, was discovered to bind the constitutive transport element (CTE) of type D retroviruses and mediate the export of unspliced viral RNAs containing a CTE (Braun et al., 1999; Gruter et al., 1998). Similar to HIV, the type D retroviruses hijack the human cellular export machinery to export their own RNAs. Unlike CRM1, however, TAP is an mRNA export receptor because injecting CTE RNA into the nucleus of Xenopus oocytes profoundly inhibits the export of cellular mRNAs (Pasquinelli et al., 1997). Importantly, co-expression of TAP and the human Mtr2p counterpart (p15) in the mex67Amtr2A yeast strain restored growth and stimulated mRNA export in vivo (Katahira et al., 1999).

Pre-mRNA Splicinq Factors Are Required for mRNA Export

Although both Mex67p and TAP have been demonstrated to crosslink in the presence of UV to poly(A) RNA in vivo, neither of these proteins bind very strongly to non-CTE RNA in vitro (Katahira et al., 1999; Segref et al., 1997). Analogous to importin a for protein import, an hnRNP-like protein (Yralp in yeast; Aly/REF in humans) serves as an export adapter, bridging the connection between mRNA and export receptors (Mex67p or TAP) (Portman et al., 1997; Strasser and Hurt, 2000; Stutz et al., 2000; Zenklusen et al., 2001) (also see Figure 3). Other mRNA export adapters have also been described in human cells (for review see Moore and Rosbash, 2001). Using cell permeable peptides to selectively inhibit the association of hnRNP Al and HuR with their respective











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36


export receptors (Trn 1 and Trn 2) Gallouzi and Steitz (2001) demonstrated that these RNA-binding proteins mediate the export of two distinct mRNAs. The Steitz group also provided evidence that SR proteins bind to ESEs and directly interact with TAP to stimulate the export of spliced mRNAs (Huang et al., 2003). In humans, several pathways may exist to export different sub-classes of mRNAs, although the primary export pathway likely involves the cooperative recruitment of TAP by REF/Aly and various SR proteins. What about yeast? Have other adapter proteins been described? To date, Yralp, and Yra2p are the only RNA-binding proteins known to functionally interact with Mex67p. Npl3p (also known as Nablp) and Gbp2p contain SR-like domains, associate with nuclear poly(A) RNA and shuttle between the nucleus and cytoplasm (Lee et al., 1996; Wilson et al., 1994; Windgassen and Krebber, 2003). Mutations in NPL3 and increased expression of GBP2 lead to nuclear accumulation of poly(A) RNA suggesting these genes may play a role in the export of mRNA (Lee et al., 1996; Windgassen and Krebber, 2003). However, a direct role for Npl3p and Gbp2p in the recruitment of export receptor(s) to the mRNA has not been demonstrated.

Surprisingly, formation of the Mtr2p-Mex67p-Yral p-mRNA export complex does not require RanGTP (Katahira et al., 1999). ATP-hydrolysis is believed to fuel association and dissociation of mRNA export complexes because several DExD/H-box helicases are required for mRNA export (reviewed in Linder and Stutz, 2001). Yralp has an affinity for the spliceosomal ATPase Sub2p (Strasser and Hurt, 2001). Importantly, Sub2p and Mex67p interact with overlapping domains of Yral p and these interactions are mutually exclusive in vitro (Strasser





37


and Hurt, 2001). These results suggest that Yralp-mRNP export takes place in two distinct steps (Figure 3). First, Yralp and Sub2p associate with pre-mRNA during transcription and splicing. Second, Sub2p is exchanged for Mex67p so that the mRNA can be released from RNAP II and targeted to the NPC by Mex67p-Mtr2p. It is important to point out that it is not clear if Sub2p ATPase activity is required for mRNA export or if Sub2p plays a structural role in recruiting Yralp to the pre-mRNA during splicing. Nevertheless, these interactions are conserved in metazoans (Luo et al., 2001) and provide a mechanism for coordinating pre-mRNA splicing with mRNA export (reviewed in Reed and Hurt, 2002). A second DExD/H-box helicase (Dbp5p) is also required for efficient export of poly(A) RNA (Snay-Hodge et al., 1998). Dbp5p exhibits RNA-dependent helicase activity in vitro (Tseng et al., 1998) and associates with Rat7p/Nupl59p on the cytoplasmic side of the nuclear envelope in vivo (Hodge et al., 1999; Schmitt et al., 1999; Strahm et al., 1999). These results have led to the proposal that Dbp5p awaits the exported mRNP on the cytoplasmic side of the nuclear pore, exchanging nuclear-associated proteins for cytoplasmic mRNA binding proteins immediately after export (Lei and Silver, 2002; Reed and Hurt, 2002).

TREX Links Transcription, Splicingq and mRNA Export

Sub2p and Yralp are both recruited during transcription by members of the THO complex (Tho2, Hprl, Mftl and Thp2) (Strasser et al., 2002). Importantly, Sub2p and Yralp associate with highly active genes during transcription elongation and temperature-sensitive mutations in any of the THO





38

genes result in nuclear accumulation of poly(A) RNA export at restrictive growth temperatures (Strasser et al., 2002; Zenklusen et al., 2002). Mutations in sub2, yral, mex67 and mtr2 also lead to defects in transcription elongation and exhibit hyper-recombination phenotypes as previously described for THO mutants (Jimeno et al., 2002). In addition, Mex67p-Mtr2p interacts genetically and physically with the Sac3p-Thplp complex (Fischer et al., 2002). THP1 is required for efficient transcription elongation (Gallardo and Aguilera, 2001) and SAC3 has an essential role in both mRNA export and progression through the cell cycle (Jones et al., 2000; Lei et al., 2003). The precise mechanisms linking transcription, splicing and mRNA export have not been fully elucidated yet, but one can suppose that THO recruits Yralp and Sub2p during transcription elongation (see Figure 3). If Yralp and Sub2p are not present, THO components inhibit elongation so that mRNA synthesis is halted in the absence of functional export factors. If Yralp and Sub2p associate with the THO components, however, efficient transcription elongation and pre-mRNA splicing proceeds without further incident. Exchanging Sub2p-Yralp interactions for Mex67pMtr2p-Yralp interactions leads to a TREX-released mRNP and interactions between Mex67p-Mtr2p and Sac3p-Thplp mediate the translocation of the mRNP to the cytoplasm (Figure 3).

Connections between Polyadenylation and mRNA Export

Splicing is unlikely to provide the only link between pre-mRNA processing and mRNA export because only a small percentage of yeast genes contain introns (Davis et al., 2000). In addition, the D. melanogaster and C. elegans





39

REF/Aly genes are dispensable for mRNA export and cell viability (Gatfield and Izaurralde, 2002; Longman et al., 2003). In contrast, UAP56 is essential for all stages of C. elegans development and RNAi experiments reveal an essential role for the helicase in mRNA export. Aside from the previously described role in splicing, Sub2p/UAP56 activity may also be required for release of mRNPs from the site of transcription after polyadenylation because mutations in sub2 lead to an accumulation of intronless RNAs at or near the site of transcription (Jensen et al., 2001a).

It has been known for several years that polyadenylation can stimulate the export of mRNA (Eckner et al., 1991; Huang and Carmichael, 1996). Mutations in components of the cleavage and polyadenylation machinery block poly(A) RNA export (Brodsky and Silver, 2000; Hammell et al., 2002; Libri et al., 2002) and mRNA export mutants accumulate hyperadenylated RNA in nuclear foci (Hilleren and Parker, 2001; Jensen et al., 2001b; Kadowaki et al., 1994; Tseng et al., 1998). Nuclear accumulation of poly(A) RNA in mRNA export mutants (rat71 and ripIA) and 3' end formation mutants (rnal5-1 or papl-1) requires the function of nuclear exosome components (Rrp6p, Rrp4p, and Mtr4p) (Hilleren et al., 2001; Libri et al., 2002). These authors envisage the exosome as a gatekeeper for mRNA export, responsible for retaining and degrading improperly cleaved or hyperadenylated RNAs in the nucleus (see Figure 4). The exosome consists of at least ten different proteins with 3' to 5' exonuclease activity (Allmang et al., 1999b; Mitchell et al., 1997) and was first described as a conserved protein complex required for processing 3' ends of several small











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nuclear RNAs including the 5.8S ribosomal RNA (rRNA), small nucleolar RNAs (snoRNAs) and U4 snRNA (Allmang et al., 1999a; Mitchell et al., 1997). More recently, it has been demonstrated that normal mRNAs are also degraded in the nucleus by the exosome (Das et al., 2003; Jacobs Anderson and Parker, 1998). Deletion of nup116 leads to the retention and RRP6-dependent degradation of normal poly(A) RNA in the nucleus (Das et al., 2003). In addition to Rrp6p, the yCBC and a 5' to 3' nuclear exonuclease (Ratlp) are required for degradation of nuclear poly(A)* RNA in the nup116A strain. These authors propose that a nuclear mRNA degradation (DRN) system, consisting of the CBC and nuclear exosome, is responsible for general mRNA turnover.

How are polyadenylated mRNAs released from the site of transcription and exported to the cytoplasm? Is there an export adapter for the poly(A) tail, analogous to Yralp, that actively recruits an export receptor to the mRNA? Does a protein bind the poly(A) tail and inhibit CBC-Rrp6p recruitment to the exosome for retention and degradation in the nucleus? Answers to these questions may provide a more complete understanding of the mechanisms regulating polyadenylation, transcript release and mRNA export.














MATERIALS AND METHODS

Yeast and Bacterial Culture Media

Yeast cells were cultured at specified temperatures in YPD media consisting of 1% Bacto-yeast extract (Fisher), 2% Bactopeptone (Fisher), 2% of dextrose (Fisher), and 2% Difco-Agar (for plates), unless otherwise indicated. For growth of the nab2ApGAL:NAB2 strain, 2% galactose (Sigma) was substituted for dextrose. Gene expression from plasmids was achieved by growing yeast cells in synthetic media (.67% Bacto-yeast nitrogen base without amino acids [Difco], supplemented with the appropriate L-amino acids [Sigma] as needed) with dextrose (SD) or galactose (SG) as previously described (Rose et al., 1990). The addition of 5-flouro-orotic acid (5-FOA) (Diagnostic Chem. Ltd.) to SD media allowed for the eviction of URA3 marked plasmids when necessary (Sikorski and Boeke, 1991). Diploid cells were induced to sporulate by growth overnight on Pre-Sporulation (Pre-Spo) plates (0.8% Bacto-yeast extract, 0.003% Bacto-peptone, 10% dextrose) followed by 2-3 days of growth on sporulation (SpoX) plates (1% KOCH3, 0.025 mg/ml ZnOCH3).

Plasmid propagation in bacteria was carried out by growth at 370 C in Luria-Bertani media (1% Bacto-tryptone, 0.5% Bacto-yeast extract, 1% NaCI, pH 7.5, and 2% Difco-agar for plates) supplemented with 100 pg/ml ampicilin (Sigma) or 40 pg/ml kanamycin (Roche) as required.



43





44


Yeast Strains and Plasmids

The yeast strains and plasmids described in this study are listed in the appendix. For preparation of full-length recombinant Nab2p, primer set MSS1131/MSS1132 was designed to amplify the NAB2 gene from L4717 genomic DNA with sequence encoding six histidine residues (His6) fused to the 3' end of the open reading frame. The PCR product was cloned into pGEX-4T-1 (Amersham Pharmaciea Biotech) between the EcoRI and Sail sites. The resulting pGST-Nab2-His6 plasmid was transformed into BL21-CodonPlus (DE3) cells (Stratagene). The expressed GST-Nab2-His6 protein was purified using standard gluatathione-Sepharose (Amersham Pharacia Biotech) and His-Bind (Novagen) chromatographic techniques.

The nab2ApPAB1NL strain was constructed by amplification of the PAB1 open reading frame from L4717 genomic DNA using MSS1261 and MSS1259 followed by insertion into Xhol-BstEll-cut pYES2/SPB4 (Invitrogen) to generate an NLS-PAB1-V5 gene fusion (pPAB1.3). NLS-PAB1-V5 was excised from pPAB1.3 by Smal-Xbal digestion and inserted into pRS315 (LEU2, CEN, [(Sikorski and Hieter, 1989)]) to generate pPAB1.4. The PAB1 5' and 3' untranslated regions (UTRs) were amplified by PCR from L4717 genomic DNA using MSS1287/MSS1263 and MSS1268/MSS1288 primer pairs, respectively. The 3' UTR PCR fragment was inserted between Xbal and Sacl sites and the 5' UTR between Sail and Smal sites of pPAB1.4 to generate the pPAB1.6 plasmid. The mutant NLS (mnl) sequence was fused to the 5' of the PAB1 ORF by PCR amplication from L4717 genomic DNA using primers MSS1262 and MSS1259.





45

The PABmni, DNA fragment was inserted into Smal-Sphl-cut pPAB1.6, resulting in pPAB1.7. The pPAB1.6 and pPAB1.7 plasmids were transformed into the nab2ApGAL::NAB2 shuttle strain (YRH201c) to create the nab2ApPAB1NL (YKN105) and nab2ApPABlmnl (YKN106) strains, respectively. Transformed cells were initially plated on SG media without leucine (SG-Leu) and then on YPD to test for nab2A suppression. For YKN105, cells were also streaked to 5FOA plates to select for the loss of pGAL::NAB2 (URA3, CEN, (Anderson, 1995)).

The NAB2 allele for tandem affinity purification (NAB2T) was constructed by amplification of the calmodulin binding peptide (CBP), tabbaco etch virus (TEV) protease site, protein A (ProtA) sequences from pBS1479 (Rigaut et al., 1999) using primers MSS811 and MSS812. The resulting PCR fragment also contained 50 nucleotides of homology to the 3' end of the NAB2 open reading frame fused in-frame to the TAP tag followed by sequence encoding the TRP1 gene and another 50 nucleotides of homology to the 3' UTR of NAB2. After transformation of the DNA fragment into L4717 cells, tryptophan auxotrophs were selected and expression of the Nab2pT fusion protein (YKN206) was confirmed by immunoblot analysis using aNab2 3F2 (1:1000).

For tandem affinity purification of the Nab2NpT and nab2-20NpT peptides, the 5' and 3' UTRs of NAB2 were amplified from L4717 genomic DNA with primer sets MSS1356/MSS1573 and MSS1384/MSS1358, respectively. Insertion of the 5' UTR fragment into Sall-Pstl-cut and the 3' UTR fragment into BamHI-Sacl-cut pRS315 yielded plasmid pNAB2.46. The TAP tag was excised from pBS1479





46

(Rigaut et al., 1999) using BamHI and Hindlll restriction enzymes and inserted into pNAB2.46 to generate pNAB2.47. Primers with sequence encoding the SV40 NLS and c-Myc epitope in-frame with the open reading frame of NAB2 (MSS1574 and MSS1575) were created and used to amplify the wild-type NES of NAB2 from L4717 genomic DNA and the mutant NES of nab2-20 from pnab220. The pNAB2.47 plasmid was digested with Pstl and BamHI and the wild-type NES (pNAB2.48) and mutant NES (pNAB2.54) fragments were inserted into the digested plasmid. The NAB2NT (YKN284) and nab2-2ONT (YKN287) strains were created by transformation of pNAB2.48 and pNAB2.54 into L4717, respectively. An immunoblot analysis of the YKN284 and YKN287 strains was performed using cNab2 3F2 to confirm expression of the tagged peptides in vivo (data not shown).

Strains expressing the AD-NT1 and AD-NT2 peptides for epitope mapping experiments were prepared by transforming pNAB2.42 and pNAB2.41 into AH109, respectively. The first N-terminal truncation of NAB2 (NT1) was amplified from L4717 genomic DNA using primer set MSS1423/MSS1444 and the second N-terminal trunction (NT2) was amplified from L4717 genomic DNA using primers MSS1423 and MSS1450. Both fragments were inserted into EcoRl-BamHI-cut pGADT7 (Clontech) to give plasmids pNAB2.42 (NT1) and pNAB2.41 (NT2).

The in vitro glutathione S-transferase pull-down experiments were performed by creating plasmids containing GST-LRR (pMEX67.5), GST-SUB2 (pSUB2.5), GST-PAB1 (pPAB1.8), GST-YRA1 (pYRA1.17), and GST-NPL3 (pGST-NAB1) gene fusions followed by transformation into BL21-CodonPlus





47

(DE3) cells for inducible expression of the fusion proteins. Briefly, the LRR of MEX67 was amplified from L4717 genomic DNA with primers MSS1661 and MSS1662 and inserted into EcoRI-Sall-cut pGEX-4T-1. The open reading frames of SUB2 and PAB1 were amplified from L4717 genomic DNA using primer sets MSS1729/MSS1730 and MSS1727/MSS1728, respectively. SUB2 and PAB1 were inserted into pGEX-4T-1 after digestion with BamHI and Xhol. Since the YRA1 gene contains a single intron, El was amplified from L4717 genomic DNA using primer set MSS1539/MSS1540 and E2 with primers MSS1541 and MSS1542. The El PCR fragment was inserted into BamHIEcoRI-digested pGEX-4T-1 to give pYRA1.16 and E2 was inserted into EcoRIXhol-cut pYRA1.16, creating pYRA1.17. The NAB2 N-terminal fragment was excised from pNAB2.41 by digestion with Ncol and BamHI and inserted into pET15b (Novagen) to create pNAB2.58. The N-terminal region of nab2-20 was amplified from pnab2-20 (Anderson, 1995) with MSS1423 and MSS1444 and inserted into EcoRI-BamHI-cut pGADT7 to create pNAB2.45. The mutant Nterminal fragment was excised from pNAB2.45 by digestion with Ncol and BamHI and inserted into pET15b to create pNAB2.59. The pNAB2.58 and pNAB2.59 plasmids were transformed into BL21-CodonPlus (DE3) cells for inducible expression of the Nab2pN and Nab2-20pN fragments, respectively.

Nucleic Acid Isolation Procedures

Yeast genomic DNA was isolated from 5 ml of logarithmically growing (OD600 = 0.5 2 U/ml) L417 cells. Cells were pelleted by centrifugation at 1,500 x g for 5 minutes. The pellet was re-suspended in 0.5 ml of cell wall digestion





48


buffer (800 mM Sorbitol, 100 mM EDTA, 30 mM DTT) and 50 pl of 5 mg/ml Zymolyase 100T (Seikagaku Corporation). Cell walls were digested for 1 hour at 370 C and spheroplasts were harvested by centrifugation at 3,000 x g for 1 minute. Spheroplasts were re-suspended in 0.5 ml of lysis buffer (50 mM Tris, 20 mM EDTA, 1% SDS) and incubated at 650 C for 30 minutes with mixing every 10 minutes. Proteins and cell debris were precipitated by incubating on ice in the presence of 1.8 M KOAC and pelleted at 40 C by centrifugation at 14,000 x g for 15 minutes. The supernatant was transferred to a fresh tube and DNA was precipitated in 50% isopropanol for 10 minutes at 240 C. The DNA was pelleted at 14,000 x g for 1 minute, washed and dried in a SpeedVac (Savant). After resuspension of the pellet in 250 p of TE buffer (10 mM Tris-HCI pH 7.5, 1 mM EDTA), RNA was digested at 370 C for 1 hour using 200 ng of RNase A. Proteins were extracted with phenol and genomic DNA was precipitated using 0.3 M sodium acetate. DNA was recovered by centrifugation at 14,000 x g, washed, dried and resuspended in 100 pl of TE. The concentration of genomic DNA was determined by measuring the absorbance of 260 nm light (OD260). Previously described methods were used to recover plasmids from yeast cells (Strathern and Higgins, 1991) and bacterial cells (Sambrook et al., 1989).

Total RNA was extracted from yeast cells using 650 C acid equilibrated phenol as previously described (Guthrie and Fink, 1991). Briefly, logarithmically growing cells were harvested by centrifugation at 2,000 x g for 5 minutes and resuspended in AE lysis buffer (50 mM NaOC3H, pH 5.3, 10 mM EDTA, 1% SDS). Proteins and cell debris were extracted from the supernatant at 650 C with AE





49


equilibrated phenol. RNA was precipitated at -700 C for 1 hour using 2 M ammonium acetate. After centrifugation, pelleted RNA was re-suspended in diethyl pyrocarbonate (DEPC) treated d2H20 and the concentration was determined by measuring the OD260.

Cell Transformation

Transformation of yeast cells was performed by treatment with lithium acetate (Ito et al., 1983). Cells were grown in YPD or SD to an OD6o0 = 0.5 U/ml. A total of 10 ml for each transformation was harvested by centrifugation at 1,500 x g for 5 minutes. Cells were washed with d2H20 and harvested again by centrifugation. The cell pellet was re-suspended in 700 pI of buffer (Tris-EDTA, 0.1 M lithium acetate, 43% polyethylene glycol molecular weight 3350) containing 5 pg of denatured calf thymus DNA and 1-5 pg of plasmid DNA. Incubation was carried out at 240 C for 30 minutes, rotating end over end. At last, cells were heat-shocked at 42' C for 15 minutes, washed, and plated on SD plates supplemented with the appropriate amino acids.

Electro-competent bacterial cells (DH5a) were transformed with plasmid DNA (1-100 ng) using an electric current generated by a BioRad GenePulser (2.5 kV, 200 Q, 25 pFd). After electro-poration and DNA uptake, cells were allowed to recover in LB media at 370 C for 1 hour. The cell suspension was plated on LB supplemented with appropriate antibiotics and incubated overnight at 370 C to allow for colony formation.





50

Yeast Genetic Manipulations

Mating, sporulation, and tetrad dissections were all performed as described (Guthrie and Fink, 1991). Haploid cells of complementing mating type (mat a and mat a) strains were patched on YPD plates at 240 C for 1-2 days. Diploids were selected on SD media containing the appropriate amino acids. Spore formation was achieved by incubating diploids on Pre-Spo plates for one day and SpoX plates for two days. Cell walls were digested for 10 minutes in 1.0 M sorbitol and 0.5 mg/ml Zymolyase 20T (Seikagaku Corporation) releasing individual spores for micromanipulation. Dissected spores were allowed to germinate and form colonies on YPD plates at 240 C unless otherwise indicated. Colonies were tested for various auxotrophic markers and growth at specified temperatures. The inability of individual spores to form colonies was also noted.

Yeast Total Cell Protein Isolation

Five OD600 units of yeast cells were harvested by centrifugation at 2,000 x g. Cells were washed once with d2H20 and re-suspended in 200 pl of 10% trichloroacetic acid (TCA) (Fisher) in a microfuge tube. Acid-washed beads were added to the cell suspension. The tubes were vortexed vigorously for 10 seconds and chilled on ice for 15 seconds. Vortexing was repeated for a total of six times. The lysate was removed from the beads and subjected to centrifugation at 14,000 x g for 10 minutes. The protein pellet was recovered after centrifugation and re-suspended in 100 pl of Laemmli cocktail (10 mM TrisHCI pH 6.8, 4% SDS, 20% glycerol, 1.3 M 1-mercaptoethanol, 4 mg/ml Bromphenol blue). The protein cocktail was neutralized with 1 M Tris-base and





51

boiled for 3 minutes prior to fractionation on 12.5% polyacrylamide-1% SDS gels, unless otherwise noted.

Fluorescence In Situ Hybridization and Cellular Immunofluorescence

In order to determine the subcellular localization of proteins and poly(A)* RNA, yeast cells were grown to an OD600 = 0.1 0.5 U/ml. After harvesting by centrifugtion at 1,000 x g for 10 minutes, cells were fixed in freshly prepared 4% formaldehyde (from EM grade paraformaldehyde) for 2 hours with gentle rocking. Fixed cells were pelleted at 800 x g for 2 minutes and washed twice with WB1 (100 mM KH2PO4, pH 6.5) and once with WB2 (100 mM KH2PO4, pH 6.5, 1.2 M Sorbitol). Cells were harvested as before and re-suspended in 1 ml of WB2 plus 30 mM P-mercaptoethanol. Spheroplast formation was achieved by the addition of 300 ng of Zymolyase 100T and incubation at room temperature for 30-45 minutes. After greater than 90% of the cells no longer contained cell walls as determined by phase-contrast microscopy (400X magnification), cells were collected by centrifugation at 2000 x g for 1 minute. Cells were washed with WB2 and attached to polylysine-coated 10-well HTC Blue Slides (Cell-Line Associated). Slides were washed once in ice-cold PBS (150 mM NaCI, 3 mM KCI, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) and dipped in 100% methanol for 5 minutes at -20o C followed by 100% acetone for 30 seconds. After three washes with PBS, slides were placed in PBS plus 0.1% Triton-X (Sigma) for 5 minutes and washed three more times with PBS. Slide wells were blocked for non-specific antibody interactions by the addition of 3 % BSA/PBS for 30 minutes at 240 C. Primary antibodies were diluted in 3% BSA/PBS (ctNab2, 1:500;





52

cPabl, 1:5000; aNopl, 1:500; aV5, 1:500; OaDig, 1:100) and added to respective wells for a 60 minute incubation at 240 C. Poly(A)* RNA was detected by fluorescence in situ hybridization (FISH) using a d(T)50-digoxigenin conjugated probe according to a previously published procedure (Amberg et al., 1992). The Alexa Fluor 488 goat anti-mouse IgG, secondary antibody was used to detect aNab2, aPabl, aV5 and aDig. For co-localization of poly(A) RNA and Noplp, the anti-Nopl A66 antibody (gift from John Aris, University of Florida, Gainesville, FL) was detected by the Alexa Fluor 555 goat anti-mouse IgG2A secondary antibody (Molecular Probes). DNA was detected by 0.5 pg/ml 4'6-diamidino-2phenylindole (DAPI). Cells were visualized using an Axiophot (Zeiss) microscope equipped with a X100 fluorescence/differential contrast objective and images were captured by either monochrome (Zeiss) or color (Spot) digital cameras.

In Vitro 3' End Processinq Assays

Standard 3'-end processing assays were performed essentially as described (Minvielle-Sebastia et al., 1997) with 2 pl of either YAS394 (pablA) or BMA64 (PAB1) cell extracts. Reconstitution assays with purified factors were performed with 0.5 pl of partially purified CF I (Mono Q fraction from CFII purification; (Minvielle-Sebastia et al., 1998)) and 0.5 pl of CPF (affinity purified from strain YSD10 expressing a TAP-tagged Fiplp fusion protein; unpublished data). Reactions were supplemented with either 25-800 ng of GST-Nab2p-His6, 50-800 ng of GST-Nab4p (Minvielle-Sebastia et al., 1998), or 50-1000 ng of purified bovine PABPN1 (gift of Elmar Wahle, University of Halle-Wittenberg,





53


Halle Saale, Germany). The polyadenylation time course experiment was performed with a 12-fold reaction mixture of YAS394 extract supplemented with 9.6 pg of GST-Nab2p-His6 and incubated at 300 C for 15 minutes without RNA substrate. Following addition of labeled transcript, aliquots of 20 pl were withdrawn at the specified times. Reactions were analyzed on 40 cm long 6% polyacrylamide-8.3 M urea gels.

Filter Bindinq Assays

Standard filter binding assays with r(A)25 (Dharmacon Research) and either GST-Nab2p-His6 or GST-His6 were performed as previously described (Wong and Lohman, 1993). Binding reactions were incubated at 300 C for 30 minutes in 20 mM Tris-HCI pH 7.4, 100 mM KCI, 2.5 mM MgCI2, 0.5 U/pl Superase-In (Ambion) with 5 pM 32P end-labeled A25 RNA and increasing amounts of purified GST-Nab2p-His6 or GST-His6 fusion proteins. Protein-RNA complexes were filtered on 0.45 micron nitrocellulose (Osmonics) and bound versus unbound RNA fractions were quantified using a Storm phosphorlmager (Molecular Dynamics). Each point on the curve represents the mean of three independent experiments.

Tandem Affinity Purification

Total protein extracts of YKN206 were prepared by growing cells at 300 C in 2.5 L of YPD medium to an OD600 = 2-4 U/ml. Cells were harvested by centrifugation and resuspended in 1 volume of Buffer A(TAP) (10 mM K-HEPES, pH 7.9, 10 mM KCI, 1.5 mM MgCI2, .5 mM DTT) with protease inhibitors (.5 mM PMSF, 2 mM Benzamidine, 1 [tM leupeptin, 2 RM pepstatin, 4 RM chymostatin,





54

2.6 pM aprotinin). Cells were lysed by two subsequent passages through a French-press at 1,200 PSI. After lysis, KCI was added to a final concentration of 200 mM and the supernatant was clarified by centrifugation at 60,000 x g for 30 minutes and 130,000 x g for 90 minutes. The supernatant was dialyzed against 1000X volume of Buffer D(TAP) (20 mM K-HEPES, pH 7.9, 50 mM KCI, .2 mM EDTA, .5 mM DTT, 20% v/v glycerol) with protease inhibitors (.5 mM PMSF, 2 mM Benzamidine). Protein extracts were added to 200 pl of IgG-Sepharose beads (Amersham) and incubated at 40 C for 2 hours. The beads were washed three times with IPP150 Buffer (10 mM Tris-CI, pH 8.0, 150 mM NaCI, .1% NP40) and then incubated with 1 ml of TEV Cleavage Buffer (10 mM Tris-CI, pH 8.0, 150 mM NaCI, .5 mM EDTA, .1% NP40, 1 mM DTT) and 100 units of TEV protease at 160 C for 2 hours. Three volumes of calmodulin binding buffer (10 mM Tris-CI, pH 8.0,150 mM NaCI, 1 mM MgOAC, 1 mM imidazole, 2 mM CaCI2, .1% NP40, 10 mM P-mercaptoethanol) and 200 pl of calmodulin affinity resin (Stratagene) was added to the TEV eluate and incubated at 40 C for 1 hour. After three washes with calmodulin binding buffer, Nab2pCBP was eluted using 800 pil of calmodulin elution buffer (10 mM Tris-CI, pH 8.0, 150 mM NaCI, 1 mM MgOAC, 1 mM imidazole, 2 mM EGTA, .1% NP40, 10 mM P-mercaptoethanol). For protein analysis, 400 pl of the calmodulin eluate was precipitated using 10% TCA, pelleted at 14,000 x g, and re-suspendend in 10 pl of Laemmli cocktail. The protein cocktail was neutralized with 1 M Tris-base and boiled for 3 minutes prior to fractionation on a 12.5% polyacrylamide-1% SDS gel. Proteins were visualized using either Coomassie blue stain (2.5 mg/L Coomassie blue dye





55

[BioRad], 50% methanol, 10% acetic acid) or silver stain (0.1% Silver Nitrate). Proteins were identified by either matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) in collaboration with John Aitchison and David Dilworth (Institute for Systems Biology, Seattle, WA) or immunoblot analysis as previously described (Anderson et al., 1993b) using the following antibodies and dilutions: aNab2 3F2 (1:1000), czNab2 1F2 (1:500), aKapl04 1D12 (1:1000), aPabl 1G1 (1:1000), aNab3 2F12 (1:250), aNab4 3H1 (1:500), oNpl3 (1:500) (gift from Pamela Silver, Harvard Medical School, Boston, MA), aMex67 (1:500) (gift from Ed Hurt, University of Heidelberg, Heidelberg, Germany), aYral (1:1000) (gift from Douglas Kellogg, University of California, Santa Cruz, CA) and horseradish peroxidase-conjugated sheep anti-mouse secondary (1:5000) (Amersham Pharmacia Biotech).

Preparation of Polyclonal Antisera and Monoclonal Antibodies

The University of Florida Interdisciplinary Center for Biotechnology Research (ICBR) hybridoma laboratory was responsible for preparation of the Nab2pTAP polyclonal antiserum and aKapl04 1D12 monoclonal antibody. Two injections (5 hg) of purified Nab2pTAP were performed on BALB/c mice (Harlow and Lane, 1988). Ten days after the second injection, a preliminary bleed was performed and dilutions (1:100, 1:250, 1:500) of the blood were tested for immunoreactivity against Nab2pT-associated proteins. The polyclonal antiserum (1:500) was used to screen a Xgt 11 expression library for recombinant yeast genes as previously described (Snyder et al., 1987). Positive plaques were purified, cloned and





56

sequenced. Identification of genes was accomplished by searching the Saccharomyces Genome Database (SGD) for matching DNA sequences.

To isolate the aKapl04 monoclonal antibody, spleen cells of the mouse were fused with SP2/O myeloma cells as previously described (Harlow and Lane, 1988). The 1D12 monoclonal antibody reacted against Xgt 11 phage expressing KAP104.

Poly(A) Tail Length Determination

Total RNA was prepared from whole cells and poly(A) tail lengths were determined as previously described (Minvielle-Sebastia et al., 1998). Briefly, total RNA (1 pg) was 3'-end labeled at 0o C for 20 hours with [32P]pCp (NEN) using 20 U of T4 RNA ligase (New England Biolabs) in a 30 pl reaction mix (50 mM HEPES pH 8.3, 5 pM ATP, 10 mM MgCI2, 3.3 mM DTT, 10% DMSO, 300 pg/ml acetylated BSA). Non-poly(A) RNA was digested at 370 C for 2 hours using RNases A (50 ng/pl) and T1 (1 U/pl) in 10 mM Tris-HCI pH 7.5 and 300 mM NaCI. The digestions were stopped by the addition of EDTA, proteins were extracted with phenol and RNA was precipitated using 2 M ammonium acetate and 5 pg of RNase-free glycogen (Boehringer Mannheim). Poly(A) tails were fractionated on 8% polyacrylamide-8.3 M urea gels and visualized by autoradiography.

In Vitro GST Pull-down Experiments

Direct interactions between the N-terminus of Nab2p and either the LRR of Mex67p, Pablp or Sub2p were demonstrated using the ProFoundM Pull-Down GST Protein:Protein Interaction Kit (Pierce Biotechnology). Induction of





57

recombinant proteins in strains carrying pNAB2.58 (EKN102), pNAB2.59 (EKN103), pMEX67.4 (EKN104), pSUB2.4 (EKN111), pPAB1.8 (EKN110), pGST-NAB1 (EKN109), pYRA1,17 (EKN114) or pGEX-4T-1 (EKN101) plasmids was achieved by adding 1 mM IPTG (Sigma) to TB+K (1.2% Bacto-tryptone, 2.4% Yeast Extract, 17 mM KH2PO4, 85 mM K2PO4, 0.4% glycerol, pH 7.0) growth media and growing cells at 370 C for 2 hours. Cells were pelleted and resuspended in 200 pl of TBS (25 mM Tris-HCI pH 7.2, 150 mM NaCI) with protease inhibitors (5 pg/ml pepstatin A, 1 pg/ml chymostatin, 1 mM Eaminocaproic acid, 1 mM p-aminobenzamidine, 1 pg/ml leupeptin, 2 pg/ml aprotinin). Cells were lysed by adding 200 pl of ProFound'" Lysis Buffer (Pierce Biotechnology) and the supernatant was clarified by centrifugation at 14,000 x g for 5 minutes. GST fusion proteins were bound to glutathione-Sepharose beads (Amersham Pharmacia Biotech) by incubating 800 pl of supernatant with 30 pl of beads at 40 C for 2 hours. After three washes with 800 pl of wash buffer (50% ProFound'TM Lysis Buffer, 12.5 mM Tris-HCI pH 7.2, 75 mM NaCI), an equal volume of either EKN102 or EKN103 cell lysate was added to GST pre-bound Sepharose beads. Binding reactions were carried out at 40 C for 3 hours. Beads were washed three times with wash buffer and proteins were eluted by boiling at 1000 C for 3 minutes in 20 pl of Leammli cocktail. One-half of each binding reaction was loaded onto a 12.5% polyacrylamide-1% SDS gel and proteins were detected using either aNab2 1F2 or aGST (gift from the University of Florida ICBR hybridoma core).





58

Phosphatase Treatment with X Phosphatase

For phosphatase treatment of Nab2p in mRNA export mutant strains, cells (5 OD600 units) were lysed by glass bead vortexing in 250 pi of lysis buffer (50 mM Tris-HCI pH 7.5, 100 mM NaCI, 10 mM imidazole, 1 mM EDTA, 0.2 mM Na3VO4, 10 mM NaF, 0.5 mM PMSF, 5 pg/ml pepstatin A, 1 pg/ml chymostatin, 1 mM E-aminocaproic acid, 1 mM p-aminobenzamidine, 1 pg/ml leupeptin, 2 pg/ml aprotinin). De-phosphorylation was performed by adding 400 units of X phosphatase (New England Bio-labs) to 5 pi of lysate according to the manufacturer's instructions. Reactions were stopped by precipitation of proteins in 10% TCA. Proteins were re-suspended in Laemmli cocktail and fractionated on 8% polyacrylamide-1% SDS gels. The Nab2p isoforms were detected by immunoblot analysis using aNab2 3F2. UV-light induced photocrosslinking was performed as described (Wilson et al., 1994).














RESULTS

Research Obiectives

Efficient and reliable gene expression in eukaryotic cells requires precise temporal and spatial coordination of many nuclear and cytoplasmic events. Nuclear events include mRNA transcription, capping, splicing, polyadenylation, and nuclear export whereas protein translation, and mRNA turnover occur in the cytoplasm. RNAP II coordinates nuclear processing events with transcription by recruiting the appropriate protein complexes to the 5' ends, introns and 3' ends of pre-mRNA (reviewed in Bentley, 2002; Hirose and Manley, 2000; Proudfoot et al., 2002; Steinmetz, 1997). In addition, proteins that bind to the 7methylguanylate cap stimulate splicing, polyadenylation and nuclear export of mRNA (Lewis and Izaurralde, 1997; Shatkin and Manley, 2000). Factors required for efficient splicing, in turn, interact with proteins required for polyadenylation and mRNA export (Lei and Silver, 2002; Luo et al., 2001; Lutz et al., 1996; Strasser and Hurt, 2001; Takagaki et al., 1996; Vagner et al., 2000) providing further evidence that nuclear processing events, from transcription to nuclear export, are intimately linked.

During transcription an abundant class of heterogeneous RNA binding proteins (hnRNPs) also associate with pre-mRNA and are believed to play a role in the coordination of pre-mRNA processing events and nuclear mRNA export by



59





60

binding to specific RNA sequences and serving as export adaptors for karyopherins (Dreyfuss et al., 2002; Dreyfuss et al., 1993; Krecic and Swanson, 1999; Lei and Silver, 2002; Nakielny and Dreyfuss, 1999). This view of hnRNPs as active participants in pre-mRNA processing, particularly mRNA export, is controversial. Reed and Magni (2002) have proposed that hnRNPs bind primarily to introns and need to be removed from the mRNA-Particle (mRNP) before export while splicing-dependent SR proteins and conserved mRNA export factors (Yralp and REF/Aly) actively recruit nuclear export receptors (Mex67p and TAP) to the mRNP. An alternative possibility is that hnRNPs act at the interface between nuclear RNA processing events and mRNA export to precisely orchestrate the dissociation of the RNA processing machinery prior to the recruitment of export receptors. The goal of my thesis research has been to test the latter hypothesis using the yeast hnRNP Nab2p.

Nab2p was first identified as a nuclear polyadenylated RNA binding protein required for vegetative growth in yeast (Anderson et al., 1993b). In addition to loss of viability, depletion of Nab2p resulted in an accumulation of hyperadenylated RNA in the nucleus (Hector, 2000; Hector et al., 2002). These phenotypes mirror those for several mRNA export mutants, including prp20, mtr2, rat7, rat8, mex67, glel, and rip1 (Forrester et al., 1992; Hilleren and Parker, 2001; Jensen et al., 2001b; Kadowaki et al., 1994; Tseng et al., 1998). Unique to NAB2, however, a mutant was isolated that uncouples the polyadenylation defect from the export block. At temperatures permissive for growth, nab2-21 cells exhibit hyperadenylated tails but do not accumulate





61


poly(A) RNA in the nucleus, whereas at temperatures restrictive for growth, poly(A) RNA export is also blocked. Interestingly, PABI was isolated as a high copy suppressor of nab2A growth defects but increased expression of Pabl p did not resolve the hyperadenylation defect and only mildly restored poly(A) RNA export to wild-type levels (Hector, 2000; Hector et al., 2002). Altogether, these results suggest Nab2p performs dual functions in vivo. First, Nab2p terminates polyadenylation, possibly analogous to PABPN1, by directly binding to the polyadenylate tail. Secondly, after binding and restricting tail length, Nab2p coordinates the release from RNAP II and serves as an adapter for the nuclear export of poly(A) RNA. The following specific aims were designed to provide evidence for this model: (1) demonstrate Nab2p, like PABPN1, binds to poly(A) RNA homopolymers and limits poly(A) tail length in vitro; (2) determine if NAB2, but not PABI, is responsible for limiting poly(A) tail lengths and promoting mRNA export in vivo; (3) identify the factors, in combination with Nab2p, involved in the nuclear export of polyadenylated mRNA.

Nab2p Binds Poly(A) RNA and Limits Poly(A) Tail Length In Vitro

The maturation of eukaryotic mRNA 3' ends first requires cleavage at specific sequences upstream of the transcriptional termination site by a group of cleavage factors, followed immediately by the addition of adenosine residues to the cleaved pre-mRNA (for reviews see Colgan and Manley, 1997; Proudfoot and O'Sullivan, 2002; Wahle and Keller, 1996; Zhao et al., 1999). While the number of adenosines added to a given mRNA can vary slightly depending on the cell type, developmental stage and growth conditions, poly(A) tails are generally





62

found to be between 200-300 nucleotides in mammals and 70-90 nucleotides in Saccharomyces cerevisiae (Wahle and Ruegsegger, 1999). When cells are depleted of Nab2p, however, poly(A) tail lengths increase dramatically up to 600 nucleotides (Hector et al., 2002).

Expression and Purification of Recombinant GST-Nab2p-His6 Protein

In order to determine if aberrant tail length regulation observed in nab2A cells is directly related to Nab2p function, a recombinant Nab2p (rNab2p) was produced and used for in vitro studies. Briefly, DNA sequence encoding a glutathione S-transferase (GST) protein was cloned 5' of the NAB2 start codon to generate a GST-Nab2p fusion product. The GST-Nab2p was expressed in E. col cells and purified using a glutathione column. Unfortunately, a majority of the proteins eluted from the glutathione column were breakdown products of Nab2p (Figure 5A, GST-Nab2p lane). In an attempt to purify full-length rNab2p away from the degradation products, six histidine (His6) residues were fused to the carboxyl terminus of GST-Nab2p to allow for an additional purification step using a nickel column. The use of both tags for purification purposes greatly increased the amount of full-length rNab2p that was recovered (Figure 5A, GST-Nab2p-His6 lane).

rNab2p Binds with Higqh Affinity to Poly(A) RNA Homopolymers

The affinity of Nab2p for poly(A) RNA was determined using a standard filter-binding assay and (rA)25 (Wong and Lohman, 1993). As shown in Figure 5B, the observed dissociation constant (Kd) was 10.5 nM (blue squares). Importantly, GST-His6 protein had little or no affinity for poly(A)Y RNA (Figure 5B,










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green circles) and bovine poly(A)-binding protein (PABPN1) has been shown previously to have an affinity for (rA)25 (Kd = 7 nM; Gorlach et al., 1994) comparable to Nab2p. Considering Nab2p binds strongly to poly(A) RNA and is localized to the nucleus of yeast cells (Anderson et al., 1993b), we reasoned that Nab2p functions to restrict poly(A) tail lengths. In vitro assays were utilized to assess the role of Nab2p in restricting poly(A) tail length during polyadenylation. Nab2p Restricts Poly(A) Tail Lenqth In Vitro

Although cleavage and polyadenylation reactions are tightly coupled in vivo, these two activities can be separated in vitro. A labeled iso-1-cytochrome c (CYC1) RNA is cleaved and polyadenylated when added to yeast whole cell protein extracts (WCE) (Butler and Platt, 1988). Remarkably, the addition of cordycepin triphosphate or substituting CTP for ATP in WCE inhibits polyadenylation but not cleavage of the precursor CYC1 RNA (Butler et al., 1990). Alternatively, if the precursor CYC1 RNA is pre-cleaved (CYC1pre) at the normal cleavage position using a convenient restriction site, this CYC1pre RNA is still polyadenylated (Lingner et al., 1991a; Lingner et al., 1991b). If pablA extracts are used, tail lengths increase to about 200 nucleotides. The addition of recombinant Pablp to these extracts restricts poly(A) tail lengths to about 60 nucleotides (Amrani et al., 1997; Minvielle-Sebastia et al., 1997). It has been demonstrated that Pablp binds to poly(A) RNA and recruits poly(A) nucleases (Pan2p and Pan3p) to the tail which degrades the poly(A) RNA in a 3' to 5' manner (Boeck et al., 1996; Brown et al., 1996). These authors propose poly(A) tails are normally synthesized very long but Pablp gradually deadenylates tails





66

through the activity of Pan2p and Pan3p (Brown and Sachs, 1998). Maximum poly(A) tail length (80-100 nucleotides) for pabI and pan mutants observed in vivo, however, does not correspond to the maximum tail length exhibited in vitro (greater than 200 nucleotides) (Brown and Sachs, 1998; Minvielle-Sebastia et al., 1997) suggesting an additional factor(s) is required for poly(A) tail length regulation in vivo. To determine if Nab2p can restrict poly(A) tail length in vitro, in collaboration with Lionel Minvielle-Sebastia (University of Bordeux, France), increasing amounts of Nab2p was added to YAS394 (pablA/rrp46A) extracts and 32P-labeled CYC1pre RNA poly(A) tail lengths were examined. Note that in the presence of Pabip (Figure 6A, PAB1 control lane), poly(A) tail lengths were restricted to about 60 nucleotides but there was relatively inefficient conversion of precursor to oligo(A) and from oligo(A) to poly(A) tails. In the pablA strain, polyadenylation was unrestricted with very long tails (greater than 200 nucleotides (Figure 6A, pablA control lane). Importantly, the addition of Nab2p (Figure 6A, 400-800 ng), restricted tail lengths to approximately 90 nucleotides. Surprisingly, the addition of low levels of Nab2p stimulated polyadenylation (note the low levels of precursor and oligo(A) RNA up to 200 ng) (Figure 6A). At 400 ng and greater, Nab2p inhibited polyadenylation. This could be due to the sequestration of a factor required for efficient polyadenylation in vitro. Indeed, Nab2p interacts with the CF IB (Nab4p) in the yeast two-hybrid system (Krecic, 1998; Uetz et al., 2000). Nab4p is required for cleavage site selection and stimulates polyadenylation in combination with CF IA and PAP (Kessler et al., 1997; Minvielle-Sebastia et al., 1998). As shown in Figure 6B, the addition of










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Nab4p to pablA extracts stimulates hyperadenylation in vitro suggesting Nab2p and Nab4p may have antagonistic functions in polyadenylation. Remarkably, the addition of low levels of bovine PABPN1 (up to 200 ng) restricts tail lengths in the yeast system while inhibiting polyadenylation at higher concentrations (greater than 400 ng) (Figure 6C).

These results indicate that Nab2p is necessary for proper tail length control in vitro using whole-cell extracts, but it was also of interest to determine if Nab2p is sufficient for tail length restriction in the absence of other cellular proteins. To accomplish this, rNab2p was added to highly purified cleavage and polyadenylation factors, CF I and CPF. As before, hyperadenylation of the CYC1pre RNA was observed in the absence of Pablp (Figure 7A) (Amrani et al., 1997; Minvielle-Sebastia et al., 1997). The addition of low levels (25-50 ng) of Nab2p increased polyadenylation efficiency and limited tails to -90 nucleotides in length while higher levels of Nab2p (100-200 ng) profoundly inhibited polyadenylation (Figure 7A). Importantly, this activity of Nab2p was not specific for the uncoupled polyadenylation reaction because using a non-cleaved CYC1 RNA Nab2p promoted polyadenylation while restricting tail lengths (Figure 7B).

The mechanism for poly(A) restriction by Pablp has been proposed to involve hyperadenylation followed by deadenylation (Brown and Sachs, 1998) whereas PABPN1 is believed to bind to the poly(A) tract during synthesis and terminate polyadenylation at a specific length (Wahle, 1995). To discriminate between these two potential models for Nab2p activity, the effect of 400 ng of Nab2p on polyadenylation in pablA extracts at various time points was











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determined (Figure 7C). Remarkably, even at the earliest time point (2 min), Nab2p restricted poly(A) tail length to 90 nucleotides. Over time (up to 180 minutes), no further decrease in poly(A) tail length was observed. These results are contrary to those obtained with 250 ng of Pablp in which poly(A) tails successively shorten over time (Minvielle-Sebastia et al., 1997) suggesting the mechanisms of poly(A) tail length regulation for Nab2p and Pablp are distinct. It is important to note that wild type extracts (Figure 6A and Figure 6C, PABI lanes) exhibit tail lengths about 20 nucleotides shorter than pablA extracts that have been augmented with 400 ng of Nab2p. This is likely to reflect the PablPan2/3 activity present in wild type extracts, but not in pablA extracts (Brown and Sachs, 1998; Brown et al., 1996). We conclude from these results that Nab2p is an essential nuclear poly(A)-binding protein required for efficient polyadenylation and poly(A) tail length restriction in vitro.

NAB2 Limits Poly(A) Tail Length and Promotes mRNA Export In Vivo

To explore the possibility that Nab2p is also responsible for poly(A) tail length restriction in vivo, a high-copy suppressor screen was previously employed to isolate proteins that could compensate for loss of Nab2p (Hector, 2000). Surprisingly, high-copy PAB1 was the only identified suppressor of the nab2A growth defect. Initially these results argued that the essential function of Nab2p is in poly(A) tail length control. Closer inspection of the nab2ApPAB1 phenotypes, however, revealed that increased PABI expression did not correlate with significantly shortened poly(A) tails (Hector, 2000; Hector et al., 2002). Poly(A) tail lengths were approximately 140 nucleotides in length in nab2ApPAB1





73

cells, a mere 20-30 nucleotides shorter than tail lengths observed in nab2ApGAL::NAB2 when grown in glucose (Hector et al., 2002). Pablp also regulates mRNA turnover (Tucker and Parker, 2000; Wilusz et al., 2001) and promotes translation initiation through interactions with the poly(A) tail and elF4E (Sachs and Davis, 1989). Thus, higher levels of Pablp in the cytoplasm may lead to increased mRNA stability and a coincident upsurge in translation initiation thereby alleviating the deleterious effects of the export block in the nab2A strain. Arguing against this explanation, Pabip was found to be present in both the nucleus and cytoplasm by immunofluorescence microscopy when PAB1 was expressed from a high-copy plasmid (Hector et al., 2002). Nuclear Targetingq of Pab lp Suppresses the nab2A Growth Defect

To prove Pablp is required in the nucleus to compensate for the loss of Nab2p, DNA encoding an SV40 nuclear localization (NL) sequence (PKKKRKV) and a mutant NL (mnl) sequence (PKTKRKV) was cloned 5' of the PAB1 start codon in a low-copy plasmid (pPAB1NL and pPAB1mni, respectively). In addition, as a means of discriminating between endogenous Pablp and the PablNL or Pablmnl fusion proteins, the stop codon of PAB1 was replaced by sequence encoding the V5 epitope (Invitrogen). These plasmids were transformed into the nab2ApGAL::NAB2 shuttle strain (YRH201c). When grown on galactose at 240 C, both the nab2ApPAB1NL and nab2ApPABlmni strains grow (Figure 8A) because Nab2p is still present (Figure 8B and data not shown). When cells were streaked to glucose, only the nab2ApPAB1NL and NAB2 strains show growth (Figure 8A). Importantly, the Pab1NL and Pablmn proteins were expressed at











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76

levels similar to the endogenous Pablp as indicated by immunoblot analysis using aPabl 1G1 (Figure 8B). Unlike endogenous Pablp, however, the PablNL protein, as visualized by cell immunofluorescence using the cV5 antibody (Invitrogen), accumulated in the nucleus (Figure 9A). Surprisingly, import of the PablNL protein was incomplete (Figure 9A) suggesting either import by the SV40 NLS in yeast is relatively inefficient or PablNL is also rapidly exported. Nevertheless, these results clearly demonstrate Pablp must cycle through the nucleus to compensate for the loss of Nab2p. Nab2p Is Required for Poly(A) Tail Lenqth Control and mRNA Export

How does nuclear Pablp suppress the growth defect of a nab2A strain? Since Pabl p has been implicated in poly(A) tail trimming in vitro and in vivo (Amrani et al., 1997; Brown et al., 1996; Minvielle-Sebastia et al., 1997), poly(A) tail lengths were assayed in nab2ApPABlNL (data not shown). Poly(A) tails were actually 10-20 nucleotides longer than previously described for nab2ApPAB1 (Hector et al., 2002) indicating, in the absence of Nab2p, targeting Pablp to the nucleus is insufficient to limit poly(A) tail length in vivo. The localization of poly(A) RNA in both the nab2ApPAB1NL and nab2ApPAB1 strains was also determined by fluorescence in situ hybridization (FISH) using a d(T)50 probe. A major portion of the poly(A)* RNA signal was present in the cytoplasm of both strains (Figure 9A, nab2ApPAB1NL and nab2ApPAB1 rows), although a significant nuclear accumulation of poly(A)* RNA was still observed (Figure 9A, compare poly(A)* RNA fluorescence in nab2ApPAB1NL to that of NAB2). Remarkably, nuclear accumulation of poly(A)Y RNA in nab2ApPAB1NL cells























Figure 9. PablNL accumulates in the nucleus of nab2ApPAB1NL. A) SV40 NLStagged Pablp and poly(A) RNA concentrate in the nucleus of a nab2A strain. The localizations of V5-tagged Pablp, poly(A)* RNA and DNA are compared in NAB2, nab2ApPAB1NL and nab2ApPABl1mni strains.
B) Suppression of low-copy pPAB1NL is restricted to 240 C. Serial tenfold dilutions of NAB2 and nab2ApPAB1NL strains were spotted on YPD plates. Plates were incubated for either 3 days (240 C and 370 C)
or 7 days (140 C).






78









A

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79

correlated with an inability to grow at high (370 C) and low (140 C) temperatures (Figure 9B). Two competing hypotheses can be proposed to explain these findings: (1) the accumulation of Pablp in the nucleus (Figure 9A) inhibits mRNA export as well as growth at temperatures other than 240 C or (2) Nab2p functions in some capacity to stimulate mRNA export and Pablp is unable to replace Nab2p in this role. I favor the latter hypothesis because the RNA localization and polyadenylation phenotypes of nab2ApPAB1NL resemble those observed in connection with mutations in known mRNA export factors. Poly(A) tail lengths were assayed in the temperature-sensitive mRNA export mutant (mex67-5) and the RanGAP temperature-sensitive mutant (mal-1). A severe hyperadenylation defect was observed in mex67-5 at the non-permissive temperature (370 C), but not the permissive temperature (240 C), while mnal-1 only exhibited a slight increase in poly(A) tail length at 370 C (Figure 10). Importantly, RNA1 is required for protein import but not mRNA export (Koepp et al., 1996), whereas MEX67 is required for efficient mRNA export but not protein transport (Segref et al., 1997). Therefore, a tight correlation between nuclear mRNA export and poly(A) tail length restriction was observed. These results parallel those described for several other mRNA export mutant strains, including mtr2, dbp5, and rat7 (Hilleren and Parker, 2001; Jensen et al., 2001b). Jensen et al. (2001) reported that hyperadenylated HSP104 transcripts accumulated in nuclear foci when rat7I cells were shifted to non-permissive temperatures (Jensen et al., 2001b). The nuclear exosome, in particular Rrp6p, is required for foci retention of both hypoadenylated SSA4 RNA in pap1-1 and hyperadenylated HSPI04 RNA in























Figure 10. Hyperadenylation and mRNA export defects correlate in mex67-5.
Poly(A) tail analysis of MEX67, mex67-5 and rnal-1 grown in YPD at either 240 C or shifted to 370 for 30 minutes. Each lane represents equal amounts of labeled RNA. The indicated size markers are
pBR322 Mspl fragments.






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rat7-1 (Hilleren et al., 2001). Intriguingly an accumulation of poly(A)* RNA in sub-nuclear foci was also observed for nab2AGAL::NAB2 when grown in glucose for 2 hours and nab2-21 when shifted to 140 C for 30 minutes (Anderson, 1995; Hector, 2000). Considering the nuclear exosome and Rrp6p is responsible for processing 3' ends of rRNA and snoRNA (Allmang et al., 1999a) and GFP-Rrp6p localizes predominately to the nucleolus (Huh et al., 2003), it is possible that hyperadenylated RNAs are trafficked to the nucleolus for retention and degraded by the exosome when Nab2p is not functional. Furthermore, Pablp may allow for growth when targeted to the nucleus in a nab2A strain by inhibiting nucleolar retention of poly(A) RNA. Providing support for this model, poly(A) RNA colocalized with an abundant nucleolar protein (Noplp) in both nab2AGAL::NAB2 and nab2-21 under non-permissive growth conditions (Figure 11) but poly(A)* RNA was observed throughout the nucleus and cytoplasm in the nab2ApPAB1NL strain (Figure 9A). Thus in each nab2 mutant strain examined, the lethal growth phenotype correlated with nucleolar localization of poly(A) RNA. Based on these results I propose that Nab2p normally functions to inhibit nucleolar retention, and possibly degradation, of polyadenylated RNA while at the same time facilitates the export of poly(A) RNA to the cytoplasm. Targeting Pablp to the nucleus inhibits nucleolar retention but does not restrict tail lengths or promote mRNA export in the absence of functional NAB2. Further evidence is needed to support the idea that Nab2p and Rrp6p have antagonistic roles in nuclear mRNA export/retention and poly(A)Y RNA stabilization/degradation, but























Figure 11. Loss of nab2 function leads to nucleolar accumulation of poly(A)'
RNA in vivo. Co-localization of poly(A) RNA (green) and Nopl p (red) in nab2ApGAL:NAB2 cells shifted to glucose for 2 hours and nab2-21 cells shifted to 140 C for 30 minutes. FISH analysis of poly(A) RNA distribution was performed using conventional immunoflourescence
microscopy. DNA was detected with DAPI.






84















poly(A)+ RNA Nopl p merge/DAPI nab2ApGAL:NAB2
glucose 2h





nab2-21
140 C





85

this model also predicts that Nab2p recruits a nuclear export receptor to the poly(A) tail which stimulates the nuclear export of mRNA.

Nab2p Associates with Factors Required for mRNA Export

Several recent studies have implicated Nab2p in mRNA export (Duncan et al., 2000; Green et al., 2003; Green et al., 2002; Hector et al., 2002). Loss of NAB2 in vivo or shifting mutant nab2 strains to non-permissive growth temperatures results in rapid accumulation of poly(A) RNA in the nucleus (Green et al., 2002; Hector et al., 2002). Intriguingly, Nab2p and poly(A) RNA co-localize near nuclear pores in a mutant tomi strain (Duncan et al., 2000) and Nab2p interacts with a NPC-associated protein (MIplp) in the yeast two-hybrid system (Green et al., 2003). The authors of these studies propose that Nab2p functionally interacts with export receptors and the NPC to mediate the export of poly(A) RNA. In addition, examination of the Nab2p domain structure revealed a nuclear export sequence (NES) near the N-terminus of Nab2p that was required for efficient poly(A) RNA export (Marfatia et al., 2003). Purification of Nab2pT-associated Proteins

The founding member of the RNA export factor (REF) family of proteins (Yralp) binds mRNA in the nucleus and forms a bridge between the site of transcription and nuclear pore through interactions with the nuclear export factor Mex67p (Portman et al., 1997; Strasser and Hurt, 2000; Stutz et al., 2000; Zenklusen et al., 2001). In metazoan organisms, REF/Aly, SR proteins and hnRNPs all serve as adapters for mRNA export, channeling mRNA through the nucleus to the NPC via interactions with TAP (Gallouzi and Steitz, 2001; Huang





86

et al., 2003; Longman et al., 2003; Luo et al., 2001; Moore and Rosbash, 2001; Zhou et al., 2000). It seemed likely that Nab2p may also promote mRNA export of poly(A) RNA in yeast by serving as an adapter protein, recruiting an export receptor to the poly(A) tail. The tandem affinity purification (TAP) protocol (Rigaut et al., 1999) was employed to purify proteins that specifically associate with Nab2p.

By virtue of homologous recombination, the wild-type NAB2 stop codon was replaced with sequences encoding a calmodulin binding protein (CBP) and protein A (ProtA) to generate the NAB2T strain (YKN206) (Figure 12A). A protein that migrated more slowly (Nab2pT) than the wild type protein (Nab2p) was detectable by immunoblot analysis using aNab2p 3F2 (Figure 12B) indicating recombination was successful. Importantly, levels of Nab2pT corresponded to that of Nab2p (Figure 12B) and growth rates of the two strains were comparable (data not shown). The dual CBP-ProtA tag allows for purification of native Nab2pT, and any associated proteins, by two successive steps (Figure 13). Briefly, whole cell extracts were prepared from YKN206 and incubated at 40 C with IgG Sepharose (Pharmacia). The ProtA peptide has a relatively high affinity for Immunoglobulin G (lgG) protein allowing for retention of Nab2Tp-associated proteins (Nab2pTAP) on the Sepharose beads. After several washes to remove nonspecific proteins, the addition of TEV released Nab2pTAP from the IgG Sepharose by cleavage between the CBP and ProtA peptides (see Figure 12A). The TEV eluate was subsequently incubated at 40 C with calmodulin affinity resin (Stratagene). In this case, the CBP tag is indispensable











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because 2 mM CaCI2 induces binding of Nab2p to the calmodulin resin and 2 mM EGTA chelates the CaCl2 releasing Nab2pT and all associated proteins (Nab2pTAP) under mild, non-denaturing, conditions. Nab2p and Kap104 Form an Abundant Complex in Yeast Extracts

The Nab2pTAP was first used to generate antibodies against the most abundant proteins. Standard procedures were performed to inject 5 [tg of purified proteins into BALB/c mice (Harlow and Lane, 1988). After an appropriate immune response, the polyclonal antiserum was used to screen a Xgtl 1 genomic expression library as previously described (Snyder et al., 1987). Seventeen phage clones positively reacted against the Nab2pTAP antiserum. Of these clones, thirteen were also immunoreactive to aNab2 3F2 and aNab2 1F2. The remaining clones were sequenced and discovered to encode for Nab2p (2 clones) and Kap104p (2 clones). Interestingly, both NAB2 clones initiated translation near the QP region of Nab2p suggesting 3F2 and 1F2 recognize sequences within the NES region of Nab2p (see Figure 14A). To explore this possibility further, two N-terminal truncations (NT1 and NT2) of Nab2p (Figure 14A) were fused to the GAL4 activation domain (AD) peptide (Clontech) (Figure 14B) and tested for 3F2 and 1F2 immunoreactivity. Monoclonal antibody aNab2 1F2 recognizes only AD-NT2 while aNab2 3F2 recognizes both AD-NT1 and ADNT2 peptides (Figure 14C), restricting the 3F2 epitope to the first 42 amino acids of Nab2p.

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(Aitchison et al., 1996). Kapl04p interacts with RG-rich domains present in both Nab2p and Nab4p and mediates import through additional interactions with GLFG-repeat containing nucleoporins (Nup116, Nupl00, Nupl45, and Nup57) (Aitchison et al., 1996). Together, RanGTP and RNA abrogate the binding of Kapl04p to Nab2p and Nab4p, providing a mechanism for Kapl04p-Nab2/4 dissociation once in the nucleus (Lee and Aitchison, 1999). In the cytoplasm, hydrolysis of RanGTP is believed to release Nab2p and Nab4p from mRNA and induce association with Kapl04p for another round of import because Kapl04p, but not Kap95p, displaces both Nab2p and Nab4p from single stranded DNA (ssDNA) cellulose (Lee and Aitchison, 1999). Nab4p is most similar to metazoan hnRNP Al and the Kapl04p homologue (Trn 1) imports hnRNP Al into the nucleus of COS7 cells (Siomi et al., 1997). Although a metazoan protein with significant similarity to Nab2p has not been described, the RGG-domain of Nab2p interacts with Trn 1 in vitro and is sufficient for nuclear import of reporter proteins in human cells (Siomi et al., 1998; Truant et al., 1998). Recently, the Trn 1-hnRNP Al heterodimer has also been implicated in the export of specific mRNAs (Gallouzi and Steitz, 2001; Moore and Rosbash, 2001). However, a direct role for the yeast Kapl04p-Nab2p complex in mRNA export is unlikely because kap104 mutants exhibit a delayed (> 3 hours) accumulation of poly(A) RNA in the nucleus (Aitchison et al., 1996). Nevertheless, discovery of Kapl04p immunoreactivty in the polyclonal antiserum suggests Kapl04p is an abundant component of the purified Nab2pTAP complex. To substantiate this claim, the




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COORDINATION OF MRNA 3’ END FORMATION AND NUCLEAR EXPORT
BY A NUCLEAR POLY(A)-BINDING PROTEIN
By
KEITH ROBERT NYKAMP
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2003

Dedicated to my grandparents, parents and brothers,
my wife, Dawn, and son, Jonas.

ACKNOWLEDGEMENTS
I would like to thank my mentor, Maurice Swanson, for providing me with
the resources and intellectual support necessary to complete my doctoral
dissertation. During my graduate studies, he has challenged me to defend my
hypotheses with unequivocal data and has equipped me with the tools needed
for a lifetime of scientific inquiry. I also thank my committee members, Alfred
Lewin, Stephen Sugrue, and Thomas Yang, for their valuable assistance at
important times throughout my graduate career. I extend a special thanks to
James Dahlberg, from the University of Wisconsin-Madison, for taking time out of
his hectic schedule to serve as my outside examiner in Gainesville. Importantly, I
thank past and present members of the Swanson Lab, in particular James
Anderson, Ron Hector, and Carl Urbinati, for their help with many of the
experiments described in this manuscript. I would also like to thank Lionel
Minvielle-Sebastia for providing the in vitro polyadenylation results and John
Aitchison for identifying purified proteins by mass spectrometry. Finally, my wife,
Dawn, deserves special mention because without her love and support none of
this would have been possible.
Ill

TABLE OF CONTENTS
ACKNOWLEDGEMENTS iii
ABSTRACT vii
INTRODUCTION 1
Integrating RNAP II Transcription and Nuclear Processing Events 6
Synthesis of Pre-mRNA 6
Transcription Elongation and Genome Maintenance 9
RNAP II Regulates Pre-mRNA Capping 10
Pre-mRNA Splicing and Recruitment of the Spliceosome 12
Regulation of Alternative Splicing by SR Proteins and hnRNPs 15
ATP-dependent Remodeling of the Spliceosome during Splicing 17
Terminal Exon Definition Requires the Cap and 3’ Cleavage Site 19
RNAP II and the Spliceosome Connect Splicing to Transcription 21
3’ End Cleavage and Polyadenylation 23
Monitoring 3’ End Integrity during Transcription Elongation 27
Coordinating Nuclear Processing Events and mRNA Export 29
General Mechanisms of Nucear/Cytoplasmic Transport 29
mRNA Export Requires a Complex Array of Factors 31
Pre-mRNA Splicing Factors Are Required for mRNA Export 33
TREX Links Transcription, Splicing and mRNA Export 37
Connections between Polyadenylation and mRNA Export 38
MATERIALS AND METHODS 43
Yeast and Bacterial Culture Media 43
Yeast Strains and Plasmids 44
Nucleic Acid Isolation Procedures 47
Cell Transformation 49
IV

Yeast Genetic Manipulations 50
Yeast Total Cell Protein Isolation 50
Fluorescence In Situ Hybridization and Cellular Immunofluorescence 51
In Vitro 3’ End Processing Assays 52
Filter Binding Assays 53
Tandem Affinity Purification 53
Preparation of Polyclonal Antisera and Monoclonal Antibodies 55
Poly(A) Tail Length Determination 56
In Vitro GST Pull-down Experiments 56
Phosphatase Treatment with X Phosphatase 58
RESULTS 59
Research Objectives 59
Nab2p Binds Poly(A) RNA and Limits Poly(A) Tail Length In Vitro 61
Expression and Purification of Recombinant GST-Nab2p-His6 Protein 62
rNab2p Binds with High Affinity to Poly(A) RNA Homopolymers 62
Nab2p Restricts Poly(A) Tail Length In Vitro 65
NAB2 Limits Poly(A) Tail Lengths and Promotes mRNA Export In Vivo 72
Nuclear Targeting of Pab1 p Suppresses the nab2A Growth Defect 73
Nab2p Is Required For Poly(A) Tail Length Control and mRNA Export.... 76
Nab2p Associates with Factors Required for mRNA Export 85
Purification of Nab2pT-associated Proteins 85
Nab2p and Kap104p Form an Abundant Complex in Yeast Extracts 89
Nab2p Co-purifies with Nuclear mRNA-binding Proteins 95
mRNA Export Is Inhibited by a Mutation in the NES of Nab2p 100
Mex67p Interacts with the N-terminus of Nab2p 101
nab2-20 Stabilizes Mex67p-Nab2p Interactions In Vitro 104
Nab2p Is Phosphorylated in Strains Defective for mRNA Export 110
DISCUSSION 115
Nab2p Is Required for Poly(A) Tail Length Restriction 115
Potential Role for Nab2p in Preventing Nucleolar Retention of mRNA 121
Nab2p Interacts with Import and Export Receptors 126
Limitations 130
V

Conclusions 132
APPENDIX 133
REFERENCES 138
BIOGRAPHICAL SKETCH 167
VI

Abstract of Dissertation Presented to the Graduate School
Of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
COORDINATION OF MRNA 3’ END FORMATION AND NUCLEAR EXPORT BY
A NUCLEAR POLY(A)-BINDING PROTEIN
By
Keith Robert Nykamp
December 2003
Chair: Maurice S. Swanson
Major: Molecular Genetics and Microbiology
Eukaryotic messenger RNA is transcribed in the nucleus and translated
into protein in the cytoplasm. Prior to export from the nucleus, mRNAs must be
capped at the 5’ end with a 7-methylguanylate (m7G), introns must be removed
via splicing, and a polyadenylate tail must be added to the 3’ end. Splicing and
polyadenylation are carried out by large macromolecular machines and are
coordinately regulated by RNA polymerase II (RNAP II) transcription. During
transcription, members of a large family of nuclear RNA-binding proteins, the
heterogeneous nuclear RNA binding proteins (hnRNPs), bind to nascent RNAP II
transcripts. Co-transcriptional association of hnRNPs with nascent pre-mRNA is
thought to regulate the specificity and timing of subsequent RNA processing
events by influencing the recruitment of the spliceosome and
cleavage/polyadenylation factors. Following pre-mRNA processing, export from

the nucleus is promoted by interactions between the mRNA and specialized
nuclear export factors.
Although hnRNPs associate with nuclear poly(A)+ RNA and are required
for efficient mRNA export in vivo, their role in mRNA export has remained
controversial. Suggestions have been made that hnRNPs bind non-specifically
to pre-mRNA and do not directly recruit mRNA export receptors. Alternatively,
hnRNPs may act at the interface between mRNA processing events and nuclear
export, orchestrating the temporal recruitment of mRNA export factors after
processing has occurred in vivo. The goal of the research presented in this
report was to test the latter hypothesis using the yeast hnRNP Nab2p. My
results demonstrate that Nab2p is a nuclear poly(A)-binding protein, required for
both termination of polyadenylation and mRNA export. Surprisingly, these two
processes can be uncoupled in nab2 mutant strains, and Nab2p interacts with
the nuclear mRNA export factor Mex67p. Based on these results, the proposition
is made that Nab2p coordinates the termination of polyadenylation with mRNA
export in vivo.
viii

INTRODUCTION
The synthesis of messenger RNA (mRNA) in a eukaryotic cell occurs in
the nucleus and involves transcription of pre-mRNA by RNA polymerase II
(RNAP II) followed by several processing steps, including capping, splicing, and
polyadenylation (see Figure 1). The translation of mRNA into proteins, however,
occurs in the cytoplasm. Importantly, nuclear pore complexes (NPCs) allow for
the translocation of RNAs and proteins through the nuclear envelope (NE)
(reviewed in Ryan and Wente, 2000).
Compartmentalization of transcription and pre-mRNA processing in the
nucleus and protein translation in the cytoplasm allows for much greater
regulation of gene expression than could be attained otherwise (reviewed in
Hood and Silver, 1999; Komeili and O'Shea, 2000). For example, p53 normally
shuttles between the nucleus and cytoplasm, but localizes to the nucleus during
stress conditions where it increases the transcription of stress response genes
(Middeler et al., 1997). Interestingly, the inability of p53 to localize in the nucleus
correlates with the proliferation of several tumors emphasizing the importance of
protein import regulation (Moll et al., 1995; Shlamp et al., 1997).
The addition of a 7-methylguanylate cap to the 5’ end and a polyadenylate
tail to the 3’ end of mRNA provides additional opportunities for the eukaryotic cell
to regulate gene expression (Shatkin and Manley, 2000). Under normal
1

2
conditions, the cytoplasmic cap-binding protein (elF4E) and poly(A) binding
protein (PABP), protect against degradation and aid in protein synthesis through
interactions with the cap and poly(A) tail, respectively (reviewed in Wilusz et al.,
2001). AU-rich element (ARE) RNA-binding proteins, such as AUF1/hnRNP D
and tristetraprolin (TTP) bypass PABP-dependent mRNA stabilization by
promoting mRNA decay when bound to 3’ untranslated (UTR) regions of proto¬
oncogene and cytokine mRNAs (Lai et al., 1999; Laroia et al., 1999; Loflin et al.,
1999). Alternatively, HuR antagonizes the binding of AUF1/hnRNP D to AREs
and augments PABP-dependent stabilization of these mRNAs (Fan and Steitz,
1998; Gallouzi et al., 2000). Other ARE-binding proteins (TIAR and TIA-1) have
been demonstrated to inhibit PABP-dependent re-initiation of protein synthesis
(Gueydan et al., 1999; Piecyk et al., 2000). Importantly, ARE mutations or
aberrant levels of ARE-binding proteins correlate with a variety of diseases,
including autoimmunity, arthritis, myeloid hyperplasia, and tumorigenesis (Gouble
et al., 2002; Kontoyiannis et al., 1999; Taylor et al., 1996). Properly regulated
mRNA turnover is clearly very important for cell viability.
Alternative pre-mRNA splicing also gives rise to extraordinary levels of
gene regulation by increasing the number of different proteins that can be
produced by a single gene (Smith and Valcarcel, 2000). The D. melanogaster
Down’s Syndrome cell adhesion molecule (Dscam) gene provides an extreme
example of this phenomenon (Schmucker et al., 2000). The authors predict that
-38,000 distinct protein isoforms are generated by alternative pre-mRNA
splicing. Given so much diversity and complexity, it is not surprising that

r
3
symptoms of many diseases have been correlated with splicing errors (reviewed
in Faustino and Cooper, 2003). For example, mutations that eliminate or alter
constitutive c/s-acting splicing signals often result in abnormal mRNAs due to
aberrant intron inclusion or exon exclusion. In most cases, these abnormal
mRNAs are susceptible to degradation by nonsense-mediated decay (NMD) and
result in lost function (Culbertson, 1999). If aberrantly spliced mRNAs are
exported, however, they may generate truncated proteins with additional,
possibly deleterious, functions.
While pre-mRNA processing events and nucleocytoplasmic transport
provide many opportunities for gene regulation, they also create logistical
problems for eukaryotic organisms. What mechanisms are in place to ensure
that only mature, and not incompletely processed, mRNAs are exported to the
cytoplasm? It turns out that proteins essential for transcription are also important
for accurate pre-mRNA processing and factors vital for pre-mRNA processing
events are necessary for efficient mRNA export (for reviews see Flirose and
Manley, 2000; Jensen et al., 2003; Maniatis and Reed, 2002; Proudfoot et al.,
2002; Steinmetz, 1997). Integrating transcription, pre-mRNA processing and
mRNA export makes perfect sense because this linkage provides a mechanism
for the eukaryotic cell to assess mRNA quality at each step of the gene
expression pathway before proceeding to the next step.

Figure 1. A model depicting nuclear and cytoplasmic events associated with gene expression in eukaryotic organisms.
Efficient synthesis and reliable processing of pre-mRNA in the nucleus requires a network of interactions
between RNAP II and pre-mRNA processing machineries. The quality of mRNA is assessed prior to export,
reducing the likelihood that improperly processed mRNAs will adversely affect protein translation in the
cytoplasm. See text for details.

Capping
Splicing
elF4E
AAAA
Translation

6
Integrating RNAP II Transcription and Nuclear Processing Events
Synthesis of Pre-mRNA
RNAP II transcribes all eukaryotic pre-mRNA and is the primary target for
gene regulation as well as the principal conductor of pre-mRNA processing
events. Importantly, the mechanisms of pre-mRNA transcription are highly
conserved among eukaryotes allowing researchers to study transcription
initiation and elongation in a variety of model organisms. Genetic screens in D.
melanogaster and S. cerevisiae, as well as biochemical strategies in human and
yeast cells, have identified a holoenzyme consisting of five major protein
complexes (RNAP II, General Transcription Factors (GTF), Srb-mediator; SWI-
SNF, and CDK) required for transcription from chromatin-associated DNA
templates (for reviews, see Conaway and Conaway, 1993; Kornberg et al.,
1992; Lemon and Tjian, 2000; Myer and Young, 1998; Woychik and Hampsey,
2002). The core enzyme consists of twelve subunits all of which are required for
enzymatic activity. In yeast, these subunits are encoded by genes RPB1 through
RPB12. An interesting feature of RNAP II is a disordered COOH-terminal repeat
domain (CTD) consisting of twenty-six consensus YSPTSPS heptad repeats
encoded by the yeast RPB1 gene (Cramer et al., 2001). The CTD is unique to
RNAP II and is central to the regulation of transcriptional initiation and elongation
(Myer and Young, 1998; Price, 2000; Woychik and Hampsey, 2002).
Before transcription is initiated and synthesis of pre-mRNA begins, the
holoenzyme assembles on the DNA at the site of transcription. RNAP II alone
does not recognize specific DNA sequences and is unable to respond to

7
transcriptional activators (Myer and Young, 1998). Promoter recognition requires
the GTF complex (TFIID, TFIIB, TFIIF, TFIIH, and TFIIE) (Roeder, 1996;
Woychik and Hampsey, 2002). DNA-binding activities associated with TFIID and
TFIIB nucleate the formation of a pre-initiation complex (PIC) immediately
upstream of the transcription start site. Addition of the remaining GTFs (TFIIF,
TFIIE, and TFIIH) completes PIC assembly on the promoter. TFIIF plays a
structural role in stabilizing PIC assembly while TFIIE and TFIIH eliminate
transcription initiation obstacles, such as supercoiled DNA and DNA-bound
nucleosomes, through ATP-dependent unwinding and remodeling activities (Dvir
et al., 2001). SWI-SNF also utilizes ATP hydrolysis to facilitate initiation and
elongation in the midst of chromatin barriers (Lemon and Tjian, 2000). Srb-
mediator receives signals from enhancer-bound activators and modulates PIC
formation and transcription initiation through interactions with RNAP II and TFIIH
(Woychik and Hampsey, 2002). Finally, the cyclin dependent kinase (CDK) and
its cyclin partner regulate PIC assembly (Liao et al., 1995). Yeast cells lacking
the CDK homologue (Srb10p) exhibit de-repression of highly repressed genes
such as SP013, GAL1, SUC2, PH05, and MFA2 (Kuchin et al., 1995; Wahi and
Johnson, 1995). Srb10p exerts its effect on transcription initiation by
phosphorylating the CTD of RNAP II on Serine 5 (Ser5) of the heptad repeats
(Hengartner et al., 1998). These findings, combined with the fact that RNAP II
purified from PIC preparations does not contain phosphates (Kang and Dahmus,
1993), suggest a model in which Srb10p suppresses transcription from a subset
of genes by blocking PIC formation. How is Srb10p activity controlled to allow for

8
transcription initiation of highly active genes? A phosphatase, Fcplp in yeast,
interacts with TFIIF and has the ability to dephosphoyrlate the CTD at both Ser5
and Serine 2 (Ser2) of the heptad repeats (Cho et al., 2001). Fcplp may
stimulate initiation by antagonizing the effects of Srb10p on Ser5. In addition,
dephosphorylation of Ser2 by Fcp1 may be required for recycling RNAP II for
another round of transcription (see below).
After PIC formation, RNAP II escapes from the promoter and enters an
early elongation phase. These steps require ATP-dependent helicase and CTD
kinase activities associated with the transcription factor TFIIH (Dvir et al., 2001).
In yeast, the product of the KIN28 gene co-purifies with TFIIH and kin28
mutations correlate with general transcription defects (Cismowski et al., 1995;
Valay et al., 1995). Hengartner et al. (1998) have further demonstrated that
Kin28p is responsible for phosphorylating Ser5 after PIC formation and may
provide a signal for elongation to commence (Hengartner et al., 1998).
Remarkably, the timing of Ser5 phosphorylation can have profound effects on
pre-mRNA transcription. Srb10p mediated phosphorylation of Ser5 prior to PIC
formation blocks transcription initiation while Ser5 phosphorylation by Kin28p
after PIC formation stimulates elongation. Clearly, promoter escape and early
elongation are highly regulated processes and these steps play an important role
in regulating gene expression (Kugel and Goodrich, 1998; Kumar et al., 1998).
For example, heat shock protein genes have stalled RNAP II complexes at their
promoters with nascent (20-40 nucleotides) transcripts waiting to elongate in
response to heat stress (Uptain et al., 1997). Defective elongation, as observed

9
in Xeroderma Pigmentosum patients, gravely affects cell viability (Liu et al.,
2001).
The phosphorylation status of Ser2 plays an important role in the transition
from early to productive elongation. A Positive Transcription Elongation Factor b
(P-TEFb) is required for the synthesis of long transcripts in mammals (Reines et
al., 1999). P-TEFb is composed of a CDK subunit (Cdk9) and several cyclins
and phosphorylates Ser2 of the CTD after initiation (Price, 2000) providing
evidence that Ser2 phosphorylation is essential for efficient elongation. In yeast,
the Cdk9 homolog (Ctklp) is not essential for vegetative growth and presumably
not required for transcription elongation (Sterner et al., 1995). Corden and
colleagues have found that Ctklp regulates transcription during heat shock and
under nutritional stress (Patturajan et al., 1999; Patturajan et al., 1998). The
authors propose that Ctklp may be recruited to specific promoters in conjunction
with RNAP II. Transcription elongation of these genes is then regulated by
Ctklp-dependent phosphorylation of Ser2. Importantly, these results provide
evidence that transcription is regulated not only before initiation begins but also
during elongation.
Transcription Elongation and Genome Maintenance
Productive elongation is a very complex process and other factors have
been identified that facilitate elongation by removing protein obstacles from the
DNA, alleviating torsional strain caused by DNA supercoiling, or by interacting
with RNAP II to suppress transcriptional pausing and arrest (Reines et al., 1999).
SWI-SNF is essential for the formation of the PIC, promoter clearance and

10
elongation of certain genes (Wolffe, 1994). FACT was purified by virtue of its
ability to facilitate transcriptional elongation from chromatin templates in vitro
using only RNAP II and GTFs (Orphanides et al., 1998). Sil (PPR2 in yeast)
directly interacts with RNAP II to activate cleavage of stalled transcript so that the
enzyme can re-extend, allowing repeated attempts at elongation. In yeast, the
TFIO complex also plays a role in elongating through transcriptional pauses
(Chavez et al., 2000). Mutations in members of the THO complex, such as
Hpr1 p and Tho2p, result in defective elongation of a lacZ reporter gene, although
elongation through several other yeast genes is unaltered (Piruat and Aguilera,
1998). Mutant hpr1 and tho2 strains are also hypersensitive to the elongation
inhibitor, 6-azauracil, indicating TFIO facilitates productive elongation through
transcriptional blocks. Interestingly, hpr1 mutants also exhibit hyper¬
recombination phenotypes (Chavez and Aguilera, 1997). The authors believe
that when transcription is hindered due to elongation impediments, such as long
DNA repeats, the transcribed region becomes highly susceptible to
recombination. Indeed, a stalled RNAP II may provide a signal to the DNA
repair machinery that a region of the genome is unstable and needs to be
repaired by homologous recombination.
RNAP II Regulates Pre-mRNA Capping
The addition of a 7-methylguanylate (m7G) to the 5’ end of a pre-mRNA is
the first step in the production of mature mRNA (for reviews see Shatkin, 1976;
Shuman, 2001). Cap addition is a three-step process carried out by three distinct
enzymes in yeast. An RNA triphosphatase (Cetlp) hydrolyzes the y-phosphate

11
from the 5’ end of the pre-mRNA and a guanyltransferase (Ceglp) links a GMP
to the resulting diphosphate end via a 5’ to 5’ phospoanhydride bond (Shibagaki
et al., 1992; Tsukamoto et al., 1997). A methyltransferase (Abdlp) rounds out
the capping process by transferring a methyl group to the guanine on position 7
(Mao et al., 1995).
It has been known for a long time that capping occurs co-transcriptionally
when nascent RNA chains are between 25 and 30 nucleotides in length (Coppola
et al., 1983; Jove and Manley, 1984). How is the mRNA capping process
coordinately regulated with transcription? Several studies have provided
evidence that all three capping enzymes selectively associate with RNAP II only
when the heptad repeats are phosphorylated at the Ser5 position (Komarnitsky et
al., 2000; McCracken et al., 1997; Rodriguez et al., 2000; Shroeder et al., 2000).
Further, Ceglp activity is dependent on the presence of Cetlp and increases
with higher levels of phosphorylation at Ser5, but not Ser2 (Cho et al., 2001; Ho
et al., 1998; Ho and Shuman, 1999). Thus, the capping apparatus is recruited by
RNAP II to the site of transcription and allosterically activated before transcription
elongation commences.
The benefits of coupling transcription and capping are not biased toward
the capping reaction. Interactions among Cetlp, Ceglp and the CTD also serve
as a checkpoint for productive elongation. Myers et al. (2002) identified the
Cet1p-Ceg1p heterodimer as a repressor of transcription elongation and re¬
initiation in vitro (Myers et al., 2002). Apparently, the transcription machinery is
unable to carry on with transcription if Abdlp is not present to methylate the

12
guanylate cap. In support of these results, Jove and Manley demonstrated
twenty years ago that inhibiting transmethylation by adding S-
adenosylhomocysteine to transcription reactions in vitro suppressed initiation
(Jove and Manley, 1982).
Proper cap formation is essential for cellular viability and has been
demonstrated to be important for mRNA stability and turnover, pre-mRNA
processing, and translation initiation (Gallie, 1998; Lewis and Izaurralde, 1997;
Shatkin, 1985; Shatkin and Manley, 2000; Tucker and Parker, 2000; Wilusz et
al., 2001). These cellular functions can be attributed to proteins that specifically
recognize the m7G cap in the nucleus and the cytoplasm (Marcotrigiano et al.,
1997; Mazza et al., 2002). In the nucleus, a heterodimeric complex of 80
(CBP80) and 20 (CBP20) kDa subunits binds the cap and stimulates splicing and
polyadenylation (Cooke and Alwine, 1996; Izaurralde et al., 1994) (also see
below). In the cytoplasm, the cap is bound by a single 25 kDa polypeptide
(elF4E) that stimulates protein translation through its interactions with the cap,
PABP and several translation initiation factors (Sonenberg, 1996).
Pre-mRNA Splicing and Recruitment of the Spliceosome
In an astonishing discovery twenty-six years ago, mRNA sequences
(exons) of eukaryotic genes were found to be interrupted throughout the DNA by
non-coding segments (introns) (Berget et al., 1977; Chow et al., 1977). Exons
are joined together in a contiguous manner via two sequential transesterification
reactions (Burge et al., 1999; Kramer, 1996; Sharp, 1994). In the first reaction,
the 2’ hydroxyl of a conserved branchpoint adenosine attacks the phosphate

13
group of the 5’ splice-acceptor site. This results in two molecules: (1) exon 1
(E1) with a free 3’ hydroxyl and (2) a lariat intermediate formed by a unique 5’ to
2’ linkage between the first nucleotide of the intron and the branchpoint
adenosine. In the second reaction, the 3’ hydroxyl of E1 attacks the 3’ splice-
donor site of exon 2 (E2) generating a ligated mRNA and a free intronic lariat.
The first transesterification reaction has been shown to occur in vitro in the
absence of proteins and ATP (Valadkhan and Manley, 2001; Valadkhan and
Manley, 2003) providing evidence that catalysis is RNA-mediated. However, five
small nuclear RNAs (U1, U2, U4, U5, U6) and approximately 100 proteins
assemble into a large, energy consuming, splicing machine (spliceosome) on the
pre-mRNA and increase the efficiency and fidelity of the transesterification
reactions both in vitro and in vivo (for reviews see Adams et al., 1996; Burge et
al., 1999; Hastings and Krainer, 2001; Kramer, 1996; Madhani and Guthrie,
1994; Staley and Guthrie, 1998; Will and Luhrmann, 2001).
Spliceosome assembly begins with the selection of a 5’ splice site and the
formation of a commitment complex (CC) (Michaud and Reed, 1991; Seraphin
and Rosbash, 1989). Commitment complex formation specifies the boundaries
between introns and exons and is possibly the most important step in the
regulation of splicing. As mentioned earlier, inappropriate splice site selection
can be very detrimental to the health of a eukaryotic cell. How are relatively
short splicing signals recognized within a sea of RNA? In yeast, CC assembly is
initiated by the formation of a duplex between U1 snRNA and a conserved
GUAUGU sequence adjacent to the 5' splice site (Zhang and Rosbash, 1999).

14
Concurrent with U1 binding, a branchpoint binding protein (BBP) binds the
branchpoint adenosine sequence (UACUAAC) (Berglund et al., 1997) and
Mud2p binds a polypyrimidine tract located between the branchpoint and 3’
splice site (AG dinucleotide) (Abovich et al., 1994). Importantly, interactions
among the U1 snRNP, BBP, and Mud2p define introns within the pre-mRNA and
provide a scaffold for the recruitment of U2 snRNP (Abovich and Rosbash, 1997)
(also see below).
In metazoan organisms, highly degenerate splice site and branchpoint
sequences amid very long introns (> 1000 nucleotides) make intron recognition
much more difficult (Berget, 1995; Reed, 1996). Berget and colleagues
proposed that vertebrate splice site recognition requires interactions between the
3’ splice site of the upstream intron and the 5’ splice site of the downstream
intron for the definition of exons (Robberson et al., 1990). In support of this
model, SR proteins, characterized by RNA recognition motifs (RRM) and
arginine/serine-rich (RS) domains, bind exonic splicing enhancers (ESEs) and
participate in CC formation (Fu, 1995; Graveley, 2000). Both SF2/ASF and
SC35 interact with the U1-70K U1 snRNP protein through RS domains (Jamison
et al., 1995; Kohtz et al., 1994; Staknis and Reed, 1994) suggesting these
proteins facilitate the early recognition of 5’ splice sites. Furthermore, U1
depletion and U1 snRNA antisense experiments have demonstrated that SR
proteins, in particular SC35, promote CC formation even in the absence of
functional U1 snRNPs (Crispino et al., 1993; Tarn and Steitz, 1994).

15
Recognition of the mammalian branchpoint region also requires
branchpoint and pyrimidine tract binding proteins, mBBP and U2 auxiliary factor
(U2AF), respectively (Abovich et al., 1994; Arning et al., 1996; Berglund et al.,
1997; Ruskin et al., 1988). U2AF is composed of two subunits, U2AF65 and
U2AF35 (Zamore and Green, 1989). U2AF65 is most similar to yeast Mud2p
and binds directly to the polypyrimidine tract and 3’ splice site region of the intron
(Abovich et al., 1994; Singh et al., 1995). U2AF35 is unique to metazoan
organisms and physically interacts with both SC35 and SF2/ASF (Fu and
Maniatis, 1992; Wu and Maniatis, 1993) providing an explanation for the
observation that both 5’ and 3’ splice sites are required for CC formation in
mammals (Michaud and Reed, 1993; Reed, 1996). Thus, SR proteins bind to
sequences within mammalian exons and bridge 3’ and 5’ splice sites thereby
facilitating exon recognition.
Regulation of Alternative Splicing by SR Proteins and hnRNPs
Given their function in 5’ splice site recognition and exon definition, it is not
surprising that SR proteins also influence the selection of alternative splice sites
(Black, 2003; Faustino and Cooper, 2003; Grabowski and Black, 2001; Smith
and Valcarcel, 2000). In addition, heterogeneous nuclear RNA binding proteins
(hnRNPs) associate with the pre-mRNA during transcription and, in several
cases, antagonize the binding of SR proteins to the pre-mRNA (Dreyfuss et al.,
2002; Dreyfuss et al., 1993; Krecic and Swanson, 1999). When one of two
identical 5’ splice sites needs to be paired with a single 3’ splice site in vitro, high
concentrations of hnRNP A1 in splicing extracts inhibits the binding of U1 snRNP

16
to both 5’ splice sites (Eperon et al., 2000). However, an ESE placed near one 5’
splice site results in the recruitment of U1 snRNP to that site and precipitates the
formation of a CC on the pre-mRNA (Eperon et al., 2000). Importantly, ESE
effects are dependent on the addition of recombinant SF2/ASF to extracts.
These findings have led to the proposal that hnRNP A1, and possibly all
hnRNPs, bind indiscriminately to pre-mRNA, block binding of sequence-specific
RNA-binding proteins (i.e. U1 snRNP and SR proteins) and inhibit the inclusion
of alternatively spliced exons (Hastings and Kramer, 2001). In support of this
model, SF2/ASF displaces hnRNP A1 from ESEs when present in extracts at
high concentrations (Eperon et al., 2000; Zhu et al., 2001). Although this model
adequately explains the experimental observations of the Krainer group, it is not
in accordance with findings reported by Black and colleagues. For example,
Rook et al. (2003) described a purine-rich ESE required for inclusion of the
neuronal-specific exon (N1) of the c-src gene (Rooke et al., 2003). UV-
crosslinking and immunoprecipitation experiments have indicated that SF2/ASF,
hnRNP A1, H and F all interact with the N1 ESE. Consistent with previous
studies, SF2/ASF stimulates splicing and hnRNP A1 represses the inclusion of
N1. However, hnRNP H and F are both components of the enhancer complex
and required for N1 inclusion (Chou et al., 1999). In addition, a C to T mutation
in exon 7 of the SMN2 gene converts the surrounding sequence into an hnRNP
A1 binding site leading to exclusion of exon 7 from SMN2 mRNA (Kashima and
Manley, 2003). Taken together, these studies provide evidence that hnRNPs are
not to be dismissed as generic RNA-binding proteins and general repressors of

17
sequence-specific RNA-binding proteins. Rather, they bind defined sequences
and perform specialized roles in the regulation of alternative splicing.
It is important to point out that very few yeast genes contain introns. Of
the genes that do yield spliced mRNAs, most have consensus 5’ splice-sites,
very few have multiple introns and only one or two genes encode alternatively
spliced mRNAs (Davis et al., 2000). This may explain why only a few hnRNP
and SR proteins have been discovered in yeast and suggests that these proteins
will play important roles in other pre-mRNA processing events, besides splicing
(Anderson et al., 1993b; Wilson et al., 1994; Windgassen and Krebber, 2003;
Zenklusen et al., 2001).
ATP-deoendent Remodeling of the Spliceosome during Splicing
After 5’ splice site recognition, the pre-spliceosome is formed by a duplex
between the U2 snRNA and branchpoint sequence (Kramer, 1996; Staley and
Guthrie, 1998). Before the U2 snRNP can bind to the branchpoint, however, two
energy-dependent, structural rearrangements must occur. First, the branchpoint
base-pairing sequence of U2 snRNA must be prepared for binding. Second,
Mud2p/U2AF65 and BBP need to be removed from the U2 snRNA pre-mRNA
binding site. In yeast, Prp5p modulates the former rearrangement and the latter
is coordinated by Sub2p (Kistler and Guthrie, 2001; Libri et al., 2001; O'Day et
al., 1996; Zhang and Green, 2001). Both of these proteins are members of the
DExD/H-box RNA helicase superfamily characterized by conserved ATPase,
helicase and RNA interaction motifs (de la Cruz et al., 1999; Tanner and Linder,
2001). Temperature-sensitive mutations in prp5 result in pre-spliceosome

18
formation defects in vitro and are lethal in combination with mutant U2 alleles
(Ruby et al., 1993; Wells and Ares Jr., 1994; Yan and Ares Jr., 1996).
Subsequent experiments have demonstrated that Prp5p is responsible for an
ATP-dependent structural change in the intact U2 snRNP prior to branchpoint
binding (O'Day et al., 1996). Furthermore, mutations that lead to misfolding of
U2 snRNA are suppressed by gain-of-function mutations in cus2 while loss-of-
function alleles of cus2 are lethal in combination with both U2 and prp5 mutations
(Perriman and Ares Jr., 2000; Yan et al., 1998). Remarkably, pre-spliceosome
assembly does not require ATP in cus2A extracts indicating that Cus2p is
responsible for rendering pre-spliceosome formation dependent on ATP
(Perriman and Ares Jr., 2000; Yan et al., 1998). The authors of these studies
propose that Cus2p binds U2 snRNA and inhibits branchpoint binding until Prp5p
removes Cus2p through ATP-dependent remodeling activities. SUB2 is
homologous to the human gene UAP56 encoding the U2AF65-interacting protein
and genetically interacts with MUD2 (Fleckner et al., 1997; Kistler and Guthrie,
2001). Although SUB2 is essential for cell viability, deletion of MUD2 allows for
growth in the absence of SUB2. Cus2p and Mud2p appear to provide ATP-
dependent checkpoints during the splicing process allowing splicing to be halted,
if necessary, through regulatory control of Prp5p and Sub2p, respectively.
Four more ATP-dependent remodeling and assembly steps are required
for the transesterification steps to proceed. The U1 snRNA interaction with the 5’
splice-site must be exchanged for a mutually exclusive interaction with the U6
snRNA (Kandels-Lewis and Seraphin, 1993; Konforti et al., 1993). The U6

19
snRNA is not recruited to the spliceosome alone. Rather, the U4/U6*U5 tri-
snRNP escorts U6 to the pre-spliceosome (Kramer, 1996). For the spliceosome
to become catalytically active, an extremely stable base-pair interaction between
U4 and U6 snRNAs must be disrupted (Nilsen, 1998). After activation of the
spliceosome, both transesterification reactions require extensive remodeling of
the pre-mRNA in order to proceed in vivo (Staley and Guthrie, 1998). Prp28
removes U1 from the 5’ splice site (Staley and Guthrie, 1999), Brr2 unwinds the
U4/U6 RNA duplex (Raghunathan and Guthrie, 1998), and Prp2 restructures the
pre-mRNA during the first transesterification reaction while Prp16 does so during
the second reaction (Kim and Lin, 1996; Schwerand Guthrie, 1991).
Terminal Exon Definition Requires the Cap and 3’ Cleavage Site
As discussed above, accurate splicing requires the efficient assembly of a
commitment complex on the pre-mRNA that distinguishes exons from introns.
According to the exon definition model, proteins bind to introns near the 5’ and 3’
splice sites and interact with ESE bound proteins to form a network of
interactions across the exon. For internal exon definition, the supporting cast of
characters include U1 snRNP, U2AF, SR proteins and hnRNPs. Two additional
exons exist in the pre-mRNA and require a different collection of proteins for their
definition. A 5’ terminal exon is defined by the 5’ cap and 5’ splice site within the
first intron and a 3’ terminal exon is demarcated by a 3’ splice site of the last
intron and a 3’ end cleavage site. In order for the exon definition model to be
correct, cap-binding proteins and 3’ end formation factors must also be required
for splicing fidelity and efficiency. In fact, an interaction between the human

20
CBC and hnRNP F has been described (Gamberi et al., 1997). HnRNP F
preferentially associates with CBC-RNA complexes rather than naked RNA and
depletion of hnRNP F from HeLa cell nuclear extracts reduces splicing efficiency
in vitro. How does the CBC-hnRNP F interaction influence splicing activity?
Black and colleagues have demonstrated that hnRNP F forms a complex with
hnRNP H and SF2/ASF on the N1 exon of the c-src pre-mRNA to promote exon
inclusion (Chou et al., 1999). Possibly, when bound to ESEs, hnRNP F and
SF2/ASF form a network of interactions to define the 5’ terminal exon: hnRNP F
interacts with CBC and SF2/ASF recruits the U1 snRNP to the 5’ proximal splice
site. Studies in yeast also indicate that connections between the CBC and U1
snRNP are necessary for delineating the cap-proximal exon. Using in vivo
reporter assays, Fortes et al. (1999) demonstrated that LUC7 is required for non¬
consensus splice site recognition and Iuc7 mutant strains fail to utilize cap-
proximal splice sites if two competing 5’ splice sites are present (Fortes et al.,
1999a). Luc7p is a U1 snRNP-associated protein with an SR-like domain and is
indispensable for CC formation in vitro as well as U1 snRNP-CBC interactions in
vivo (Fortes et al., 1999a). In a second study by the same group, cell growth was
compromised in a mud2A strain if both cbp80 and cbp20 were also deleted from
the genome (Fortes et al., 1999b). Importantly, growth is only mildly affected in
either mud2A or cbp80Acbp20A strains.
For 3’ terminal exon definition, an interaction between U2AF65 and
poly(A) polymerase (PAP) in HeLa cells has been described (Vagner et al.,
2000). PAP stimulates the binding of U2AF65 to the pyrimidine tract of the

21
terminal intron. These interactions appear to be specific for metazoan organisms
because no interactions between Mud2p and yeast PAP have been described.
Furthermore, Dower and Rosbash (2002) observed efficient splicing in vivo in the
absence of both pre-mRNA capping and polyadenylation. Since yeast introns
are small, splicing signals are highly conserved, and most introns are near the 5’
end of the gene (Davis et al., 2000), interactions between the spliceosome and 3’
end formation complex may not be essential for efficient splicing in yeast. In fact
CBC-spliceosome interactions only appear to be essential when non-consensus
signals are utilized lending credence to the idea that exon definition is critical for
recognition of weak, alternatively used splice sites, such as those primarily found
in metazoan organisms.
RNAP II and the Spliceosome Connect Splicing to Transcription
Considering synthesis of pre-mRNA is carried out solely by RNAP II, it is
not surprising that RNAP II has been reported to interact with components of the
spliceosome. For example, reporter genes containing introns are not spliced in
metazoan cells if transcribed by RNAP I or RNAP III (Sisodia et al., 1987; Smale
and Tjian, 1985). Early in situ studies using Chironimus tentans Balbiani ring
genes to visualize pre-mRNA processing events during transcription discovered
that genes were spliced concurrently with transcription (Bauren and Wieslander,
1994; Kiseleva et al., 1994). In addition, phosporylated RNAP II (RNAP IIO)
increases the efficiency of splicing both in vivo and in vitro (Fong and Bentley,
2001; Fong et al., 2003; McCracken et al., 1997; Misteli and Spector, 1999).
Alternatively, the addition of hypophosphorylated RNAP II (RNAP IIA) inhibits

22
splicing in vitro (Hirose et al., 1999; Zeng and Berget, 2000). In yeast, Prp40p is
recruited to RNAP HO, but not RNAP IIA (Morris and Greenleaf, 2000). Prp40p
is a component of the U1 snRNP and directly interacts with BBP (Abovich and
Rosbash, 1997). During transcription elongation, the U1 snRNP-Prp40p complex
is tethered to RNAP II increasing the likelihood of 5’ splice site recognition.
Interactions between RNAP IIO and U1 snRNP also serve to retain the 5’ splice
site near elongating RNAP II, allowing branchpoint and 3’ splice site sequences
to be recognized by BBP and Mud2p immediately after synthesis. After binding
to the pre-mRNA, U1 snRNP, Mud2p and BBP precipitate the formation of a
commitment complex.
Interactions between RNAP II and the spliceosome also influence
transcription re-initiation and elongation. TFIIH interacts with U1 snRNA and
promoter-proximal 5’ splice sites stimulate transcription re-initiation in a in¬
dependent manner (Kwek et al., 2002). Two factors required for efficient
elongation, Sll and TAT-SF1, also co-purify with U2 snRNP and RNAP IIO in vivo
(Fong and Zhou, 2001; Robert et al., 2002). TAT-SF1 stimulates transcription
elongation in vitro through interactions with P-TEFb (Price, 2000). In yeast, the
TAT-SF1 homolog (CUS2) plays a mechanistic role in pre-spliceosome formation
(Yan et al., 1998). Altogether, these findings provide for an appealing model in
which Cus2p/TAT-SF1 and U2 snRNP are recruited to RNAP IIO and stimulate
transcription elongation most likely through interactions with Ctk1p/P-TEFb.
Importantly, Cus2p/TAT-SF1 may inhibit association of U2 snRNP with the pre-
mRNA until BBP and Mud2p/U2A65 bind to the branchpoint region of the intron.

23
Assuming commitment complex formation occurs without difficulty, Prp5p/hPRP5
remodels the U2 snRNP by removing Cus2p/TAT-SF1 and Sub2p/UAP56
dissociates Mud2p/U2A65 from the branchpoint allowing for assembly of the pre-
spliceosome.
3’ End Cleavage and Polvadenylation
Termination of RNAP II transcription coincides with cleavage of the pre-
mRNA at sequences downstream of the translation stop codon. Cleavage is
followed by the addition of a polyadenylate tail of 70-90 nucleotides in yeast and
200-300 nucleotides in mammals (Proudfoot et al., 2002; Zhao et al., 1999). Like
splicing, large protein complexes execute the cleavage and polyadenylation
reactions with precision (Colgan and Manley, 1997; Wahle and Keller, 1996).
Much has been learned about the factors responsible for cleavage and
polyadenylation because many of these proteins are conserved from yeast to
humans (see Figure 2) (Keller and Minvielle-Sebastia, 1997; Manley and
Takagaki, 1996). In yeast, an A/U-rich Efficiency Element (EE) and an A-rich
Positioning Element (PE) found upstream of the cleavage site recruit Cleavage
Factor IB (CF IB) to the pre-mRNA. A second factor, Cleavage and
Polyadenylation Factor I (CPF) associates with sequences surrounding the
Pyrimidine-Adenosine (PyA) cleavage site (Proudfoot and O'Sullivan, 2002;
Wahle and Ruegsegger, 1999). Interestingly CF IB is a single subunit factor
composed of the yeast hnRNP Nab4p/Hrp1p (Kessler et al., 1997). Nab4p
controls cleavage site selection in vitro and in vivo and increases the efficiency of
polyadenylation in vitro by interacting with the EE and recruiting CF IA and CPF

Figure 2. Mechanism of 3’ end formation is conserved from yeast to mammals. Cleavage of pre-mRNA requires ex¬
acting sequence elements and several large multi-subunit complexes. CPSF, CstF, and CF I in mammals, or
CPF, CF IA and CF IB in yeast, position the cleavage complex and recruit endonuclease activities associated
with CF II in each case. In mammals, polyadenylation is carried out by a conserved poly(A) polymerase and
regulated by interactions between the poly(A) tail and CPSF. Less is known about poly(A) tail length regulation
in yeast. See text for details.

Mammals
Yeast
M
cn

26
to the mRNA (Chen and Hyman, 1998; Gross and Moore, 2001; Minvielle-
Sebastia et al., 1998; Valentini et al., 1999). Thus, analogous to their role In
selection of alternative splice sites, hnRNPs also control the fidelity of 3’ end
cleavage site selection. After CF IB, CF IA and CPF bind to the pre-mRNA,
cleavage is carried out by an unknown endonuclease (CF II) providing a free 3’
end for the addition of adenosines by Paplp (Wahle and Ruegsegger, 1999).
Poly(A) addition in mammalian cells is initially a slow process with PAP
dissociating from the growing tail after the addition of each nucleotide. After 10
or more adenylate residues have been added, however, a nuclear poly(A)-
binding protein (PABPN1, formerly PAB II) interacts with the nascent poly(A) tail,
the cleavage and polyadenylation specificity factor (CPSF), and PAP to form a
quaternary structure which transforms PAP into a processive enzyme (Bienroth
et al., 1993; Murthy and Manley, 1995; Wahle, 1991). After approximately 200
nucleotides, the interaction between PABPN1 and CPSF is disrupted, thus
terminating the polyadenylation reaction (Wahle, 1995). What is the mechanism
of CPSF-PABPN1-PAP complex disruption? Using electron microscopy (EM), an
oligomeric PABPN1 particle has been observed to co-localize with the growing
poly(A) tail (Keller et al., 2000). Attractively, the maximum diameter observed for
a PABPN1 particle (~21 nm) correlates with the particle size of a PABPN1-
poly(A) complex when the poly(A) molecule is 200-300 nucleotides in length.
Longer poly(A) molecules do not result in larger PABPN1 particles suggesting
the maximum PABPN1 particle size restricts poly(A) tail lengths to 300
nucleotides in vivo. These authors propose a model whereby PABPN1 initially

27
converts PAP into a processive enzyme via interactions with CPSF, but these
interactions are not feasible when the PABPN1 particle reaches a maximum size,
thus converting PAP back into a distributive enzyme.
In yeast, an essential poly(A)-binding protein (Pablp) has also been
described, although Pablp shares minimal sequence similarity with PABPN1
(Sachs et al., 1986; Wahle, 1991). Pablp interacts in the yeast two-hybrid
system with the CF IA component Rna15p (Amrani et al., 1997) and recombinant
Pablp limits poly(A) tail lengths in vitro (Minvielle-Sebastia et al., 1997). The role
of Pablp in poly(A) tail length control in vivo, however, is questionable because
the steady state distribution of Pablp is cytoplasmic (Anderson et al., 1993a).
Furthermore, the examination of poly(A) tail lengths of total RNA in pab1A cells
reveals modest increases (~40 nucleotides) in vivo, whereas precursor RNAs
have poly(A) tails greater than 200 nucleotides in pab1A extracts (Minvielle-
Sebastia et al., 1997; Sachs and Davis, 1989). Together, these results suggest
that poly(A) tail length control in vivo is likely to require additional factors.
Monitoring 3’ End Integrity during Transcription Elongation
Flow are transcription and 3' end processing events coupled in vivo? The
same general theme holds true for 3’ end processing as described previously for
capping and splicing. The CTD of mammalian RNAP II is essential for efficient
cleavage at poly(A) sites and yeast strains containing CTD truncation mutants
exhibit reduced cleavage efficiency (Flirose and Manley, 1998; Licatalosi et al.,
2002; McCracken et al., 1997; Ryan et al., 2002). Components of the cleavage
and polyadenylation machinery in yeast, including Rna15p of CF IA and

28
Yhh 1 p/Cft1 p of CPF, co-purify with RNAP IIO in vivo and directly interact with the
phosphorylated CTD in vitro (Barilla et al., 2001; Dichtl et al., 2002b).
A conserved network of interactions between several GTFs and factors
involved in cleavage and polyadenylation also exists to link transcription to 3’ end
formation. Dantonel et al. (1997) immunopurified CPSF with TFIID from HeLa
cells. After transcription initiates, however, CPSF dissociates from TFIID and
remains associated with RNAP IIO. In addition, PC4, a mammalian transcription
co-activator, binds double-stranded DNA, interacts with TFIIA and RNAP IIO, and
is phosphorylated by TFIIH in vitro (Malik et al., 1998; Werten et al., 1999). PC4
also associates with the 64 kDa subunit of CstF suggesting that CstF is recruited
to the PIC as well (Calvo and Manley, 2001). These interactions have been
conserved throughout evolution because the yeast homologue of PC4 (Sublp)
binds to the CstF-64 counterpart (Rna15p) in vitro (Calvo and Manley, 2001).
SUB1 and SSU72 interact with TFIIB to assist in PIC assembly (Sun and
Hampsey, 1996). Interestingly, mutations in these genes disrupt the fidelity of
start site selection (Wu et al., 1999). Ssu72p also associates with two subunits
of CPF (Ptalp and Ydh1p/Cft2p) and is required for CPF-dependent cleavage of
the 3’ end (Dichtl et al., 2002a; Fie et al., 2003). Together, Ssu72p and Sublp
provide transcription start site specificity for RNAP II and recruit CF IA and CPF
to the site of transcription. In addition, the presence of CPF at the site of
transcription may provide a signal for RNAP II to begin elongation because Ptalp
also interacts with Kin28p (Rodriguez et al., 2000). Phosphorylation of the CTD
at Ser5 by Kin28p triggers the commencement of transcription elongation

29
(Hengartner et al., 1998). In the absence of efficient cleavage and
polyadenylation by CPF, Kin28p may not be able to phosphorylate RNAP II,
thereby halting transcription elongation. Thus, Ssu72p and Sublp ensure that
newly synthesized mRNAs are correctly cleaved and polyadenylated
Coordinating Nuclear Processing Events and mRNA Export
General Mechanisms of Nuclear/Cytoplasmic Transport
After the 3’ end has been cleaved and a poly(A) tail has been added the
mature mRNA is ready for export. How are molecules transported into and out of
the nucleus? The current model for general nuclear transport proposes that an
adapter binds both cargo and a receptor. Several adapters and receptors have
been identified in a number of different organisms that also associate with
integral NPC proteins, or nucleoporins (Nups) (reviewed in Gorlich and Kutay,
1999; Komeili and O'Shea, 2001; Nakielny and Dreyfuss, 1999; Ohno et al.,
1998; Pemberton et al., 1998). In addition to adapters and receptors, protein
transport requires the GTPase Ran (Kuersten et al., 2001). Typically for protein
import, an adapter (importin a) binds to a nuclear localization sequence (NLS)
exemplified by the single SV40 large T antigen (PKKKRKV) or bipartite
nucleoplasmin (KRPAAIKKAGQAKKKK) sequences and forms a quaternary
structure with an import receptor (importin (3), the NLS-containing protein, and
RanGDP (Gorlich and Mattaj, 1996; Weis, 1998). A cytoplasmic GTPase
Activating Protein (RanGAP) facilitates hydrolysis of GTP by Ran ensuring that
RanGDP molecules accumulate in the cytoplasm (reviewed in Azuma and
Dasso, 2000; Kuersten et al., 2001). Removal of GDP from Ran, in turn, requires

30
a nuclear guanine exchange factor (RanGEF) resulting in the accumulation of
RanGTP in the nucleus. Assembly of an import complex is strengthened in the
cytoplasm by RanGDP and recruited to the NPC by interactions between
importin (3 and nucleoporins. After translocation through the nuclear pore,
exchange of GDP for GTP in the nucleus releases the imported NLS protein from
importin a, importin (3, and Ran (Ohno et al., 1998). Although several importin a
and importin (3 proteins have been identified in metazoan organisms, the yeast
genome encodes one importin a (Srp1) and one importin |3 (Kap95) (Gorlich and
Kutay, 1999). Kap95p physically interacts with Nup116p and co-localizes with
Srplp near the NPC in vivo (lovine et al., 1995; Koepp et al., 1996). Proper
localization of Kap95p, Srplp, and NLS-containing proteins require a functional
GTPase activating protein (RnalpJ and a functional guanine exchange factor
(Prp20p) (Koepp et al., 1996).
Similar principles apply for export of proteins from the nucleus. Co¬
precipitation experiments first identified a human nuclear factor, homologous to
importin (3, associated with NUP214/CAN and NUP88 (Fornerod et al., 1997b).
The human gene was named hCRM1 because, in addition to having similarity to
importin (3, the open reading frame was homologous to a previously identified
gene in Schizosaccharomyces pombe (CRM1+). Early studies of CRM In-
indicated that it encoded a nuclear protein required for efficient DNA, RNA, and
protein synthesis and condensation of chromosomal regions was observed in
CRM1+ mutants at non-permissive temperatures (Adachi and Yanagida, 1989).
Increased expression of CRM1+ was subsequently found to confer resistance to

31
leptomycin B (LMB), an agent known to induce cell cycle arrest at G1 and G2
phases of the cell cycle (Nishi et al., 1994). Analogous to protein import, a
leucine-rich nuclear export sequence (NES) was discovered in the HIV-1 Rev
and kinase A inhibitor PKI proteins (Fischer et al., 1995; Wen et al., 1995). In
Xenopus oocytes and permeabilized HeLa cells, CRM1 has been shown to
mediate the export of reporter proteins containing an NES while LMB strongly
inhibited export of NES peptides (Fornerod et al., 1997a; Fukada et al., 1997;
Ossareh-Nazzari et al., 1997). CRM1 exhibited cooperative interactions in vitro
with both NES peptides and RanGTP providing evidence that CRM1 behaves
like a traditional transport receptor (Fornerod et al., 1997a).
mRNA Export Requires a Complex Array of Factors
In the same study, Fornerod et al. (1997) demonstrated that U snRNA
export, but not mRNA export, was stimulated in Xenopus oocytes by increased
levels of CRM1 and inhibited by LMB. In metazoan organisms U snRNAs are
exported to the cytoplasm after transcription, assembled into functional snRNPs,
and imported into the nucleus (reviewed in Paushkin et al., 2002; Will and
Luhrmann, 2001). Four of the five spliceosomal U snRNAs (U1, U2, U4, and U5)
are transcribed by RNAP II and have a m7G-cap to which the nuclear CBC is
able to bind. In addition to CRM1, the CBC also plays an important role in the
export U snRNAs (Hamm and Mattaj, 1990; Jarmolowski et al., 1994). In an
elegant study, the Mattaj group provided the final piece in the puzzle of U snRNA
export by identifying an adaptor protein (PHAX) which has the ability to bridge
CRM1 and CBC interactions (Ohno et al., 2000). PHAX contains a leucine-rich

32
NES and formation of an snRNA-CBC-PHAX-CRM1 export complex required
RanGTP. Remarkably, PHAX was unable to bind to CRM1, even in the
presence of U snRNP, CBC, and RanGTP proteins, if it was not phosphorylated.
These results provided evidence for an additional level of regulation for U snRNP
export.
At the same time CRM1 was discovered in metazoans, Stade et al. (1997)
reported that Crmlp was responsible for the export of NES peptides in yeast.
Intriguingly, these authors also observed a block to poly(A)+ RNA export in a
conditional crm1 mutant strain suggesting that CRM1 may serve as a receptor for
mRNA export in yeast. A subsequent study by Neville and Rosbash (1999),
however, demonstrated that a strain with an LMB-sensitive Crmlp continued to
synthesize proteins in the presence of LMB arguing against a direct role for
Crmlp in mRNA export. The discrepancy between these two reports can be
explained by the finding that increased expression of a cytoplasmic DExD/H-box
helicase (DBP5), required for proper poly(A)+ RNA localization, suppresses the
crm 1-dependent mRNA export block (Hodge et al., 1999). Thus, mutations in
CRM1 may indirectly affect mRNA export by altering the sub-cellular localization
of cytoplasmic mRNA export factors.
What are the receptors and adapters for mRNA export? A synthetic lethal
screen using a nup85 mutant allele identified MEX67 as a gene required for
mRNA export in yeast (Segref et al., 1997). Mex67p interacts with Mtr2p to form
a heterodimer localized to the nuclear periphery in vivo (Santos-Rosa et al.,
1998). In addition to Nup85p, Mex67p-Mtr2p physically associates with several

33
other nucleoporins in vitro and mutations in mtr2 profoundly affect the localization
of both Mex67p and poly(A) RNA in vivo (Santos-Rosa et al., 1998; Strasser et
al., 2000). A human protein (TAP), with similarity to Mex67p, was discovered to
bind the constitutive transport element (CTE) of type D retroviruses and mediate
the export of unspliced viral RNAs containing a CTE (Braun et al., 1999; Gruter
et al., 1998). Similar to HIV, the type D retroviruses hijack the human cellular
export machinery to export their own RNAs. Unlike CRM1, however, TAP is an
mRNA export receptor because injecting CTE RNA into the nucleus of Xenopus
oocytes profoundly inhibits the export of cellular mRNAs (Pasquinelli et al.,
1997). Importantly, co-expression of TAP and the human Mtr2p counterpart
(p15) in the mex67Amtr2A yeast strain restored growth and stimulated mRNA
export in vivo (Katahira et al., 1999).
Pre-mRNA Splicing Factors Are Required for mRNA Export
Although both Mex67p and TAP have been demonstrated to crosslink in
the presence of UV to poly(A)+ RNA in vivo, neither of these proteins bind very
strongly to non-CTE RNA in vitro (Katahira et al., 1999; Segref et al., 1997).
Analogous to importin a for protein import, an hnRNP-like protein (Yralp in
yeast; Aly/REF in humans) serves as an export adapter, bridging the connection
between mRNA and export receptors (Mex67p or TAP) (Portman et al., 1997;
Strasser and Hurt, 2000; Stutz et al., 2000; Zenklusen et al., 2001) (also see
Figure 3). Other mRNA export adapters have also been described in human
cells (for review see Moore and Rosbash, 2001). Using cell permeable peptides
to selectively inhibit the association of hnRNP A1 and HuR with their respective

Figure 3. A model for mRNA export in yeast. Recruitment of mRNA export adaptors to the pre-mRNA occurs during
transcription by the conserved TREX complex. Chromatin release and export of the processed mRNA is
facilitated by the exchange of TREX components for mRNA export receptors. Interactions between the export
receptors and nuclear pore complex proteins recruit mRNA to the nuclear pore. Conserved ATPase proteins
stimulate dissociation of nuclear export complexes once transported to the cytoplasm.

TREX complex
CBP80
CBP20
3' end processing
factors
Chromatin release
fi"
Paplp
AAAAAAAAA
CID
RNA polymerase II
mRNA export
complex
Export
Nucleus
ex67p
Cytoplasm
Mtr2p
Sac
159p
ra1p
CO
(Jl

36
export receptors (Trn 1 and Trn 2) Gallouzi and Steitz (2001) demonstrated that
these RNA-binding proteins mediate the export of two distinct mRNAs. The
Steitz group also provided evidence that SR proteins bind to ESEs and directly
interact with TAP to stimulate the export of spliced mRNAs (Huang et al., 2003).
In humans, several pathways may exist to export different sub-classes of
mRNAs, although the primary export pathway likely involves the cooperative
recruitment of TAP by REF/Aly and various SR proteins. What about yeast?
Have other adapter proteins been described? To date, Yralp, and Yra2p are the
only RNA-binding proteins known to functionally interact with Mex67p. Npl3p
(also known as Nablp) and Gbp2p contain SR-like domains, associate with
nuclear poly(A)+ RNA and shuttle between the nucleus and cytoplasm (Lee et al.,
1996; Wilson et al., 1994; Windgassen and Krebber, 2003). Mutations in NPL3
and increased expression of GBP2 lead to nuclear accumulation of poly(A)+ RNA
suggesting these genes may play a role in the export of mRNA (Lee et al., 1996;
Windgassen and Krebber, 2003). However, a direct role for Npl3p and Gbp2p in
the recruitment of export receptor(s) to the mRNA has not been demonstrated.
Surprisingly, formation of the Mtr2p-Mex67p-Yra1p-mRNA export complex
does not require RanGTP (Katahira et al., 1999). ATP-hydrolysis is believed to
fuel association and dissociation of mRNA export complexes because several
DExD/H-box helicases are required for mRNA export (reviewed in Linder and
Stutz, 2001). Yralp has an affinity for the spliceosomal ATPase Sub2p (Strasser
and Hurt, 2001). Importantly, Sub2p and Mex67p interact with overlapping
domains of Yralp and these interactions are mutually exclusive in vitro (Strasser

37
and Hurt, 2001). These results suggest that Yra1 p-mRNP export takes place in
two distinct steps (Figure 3). First, Yralp and Sub2p associate with pre-mRNA
during transcription and splicing. Second, Sub2p is exchanged for Mex67p so
that the mRNA can be released from RNAP II and targeted to the NPC by
Mex67p-Mtr2p. It is important to point out that it is not clear if Sub2p ATPase
activity is required for mRNA export or if Sub2p plays a structural role in
recruiting Yralp to the pre-mRNA during splicing. Nevertheless, these
interactions are conserved in metazoans (Luo et al., 2001) and provide a
mechanism for coordinating pre-mRNA splicing with mRNA export (reviewed in
Reed and Hurt, 2002). A second DExD/H-box helicase (Dbp5p) is also required
for efficient export of poly(A)+ RNA (Snay-Hodge et al., 1998). Dbp5p exhibits
RNA-dependent helicase activity in vitro (Tseng et al., 1998) and associates with
Rat7p/Nup159p on the cytoplasmic side of the nuclear envelope in vivo (Hodge
et al., 1999; Schmitt et al., 1999; Strahm et al., 1999). These results have led to
the proposal that Dbp5p awaits the exported mRNP on the cytoplasmic side of
the nuclear pore, exchanging nuclear-associated proteins for cytoplasmic mRNA
binding proteins immediately after export (Lei and Silver, 2002; Reed and Hurt,
2002).
TREX Links Transcription, Splicing and mRNA Export
Sub2p and Yralp are both recruited during transcription by members of
the THO complex (Tho2, Hpr1, Mft1 and Thp2) (Strasser et al., 2002).
Importantly, Sub2p and Yralp associate with highly active genes during
transcription elongation and temperature-sensitive mutations in any of the THO

38
genes result in nuclear accumulation of poly(A)+ RNA export at restrictive growth
temperatures (Strasser et al., 2002; Zenklusen et al., 2002). Mutations in sub2,
yra1, mex67 and mtr2 also lead to defects in transcription elongation and exhibit
hyper-recombination phenotypes as previously described for THO mutants
(Jimeno et al., 2002). In addition, Mex67p-Mtr2p interacts genetically and
physically with the Sac3p-Thp1p complex (Fischer et al., 2002). THP1 is
required for efficient transcription elongation (Gallardo and Aguilera, 2001) and
SAC3 has an essential role in both mRNA export and progression through the
cell cycle (Jones et al., 2000; Lei et al., 2003). The precise mechanisms linking
transcription, splicing and mRNA export have not been fully elucidated yet, but
one can suppose that THO recruits Yralp and Sub2p during transcription
elongation (see Figure 3). If Yralp and Sub2p are not present, THO components
inhibit elongation so that mRNA synthesis is halted in the absence of functional
export factors. If Yralp and Sub2p associate with the THO components,
however, efficient transcription elongation and pre-mRNA splicing proceeds
without further incident. Exchanging Sub2p-Yra1p interactions for Mex67p-
Mtr2p-Yra1p interactions leads to a TREX-released mRNP and interactions
between Mex67p-Mtr2p and Sac3p-Thp1p mediate the translocation of the
mRNP to the cytoplasm (Figure 3).
Connections between Polyadenylation and mRNA Export
Splicing is unlikely to provide the only link between pre-mRNA processing
and mRNA export because only a small percentage of yeast genes contain
introns (Davis et al., 2000). In addition, the D. melanogaster and C. elegans

39
REF/Aly genes are dispensable for mRNA export and cell viability (Gatfield and
Izaurralde, 2002; Longman et al., 2003). In contrast, UAP56 is essential for all
stages of C. elegans development and RNAi experiments reveal an essential role
for the helicase in mRNA export. Aside from the previously described role in
splicing, Sub2p/UAP56 activity may also be required for release of mRNPs from
the site of transcription after polyadenylation because mutations in sub2 lead to
an accumulation of intronless RNAs at or near the site of transcription (Jensen et
al., 2001a).
It has been known for several years that polyadenylation can stimulate the
export of mRNA (Eckner et al., 1991; Huang and Carmichael, 1996). Mutations
in components of the cleavage and polyadenylation machinery block poly(A)+
RNA export (Brodsky and Silver, 2000; Hammell et al., 2002; Libri et al., 2002)
and mRNA export mutants accumulate hyperadenylated RNA in nuclear foci
(Hilleren and Parker, 2001; Jensen et al., 2001b; Kadowaki et al., 1994; Tseng et
al., 1998). Nuclear accumulation of poly(A)+ RNA in mRNA export mutants (rat7-
1 and rip1A) and 3’ end formation mutants (rna15-1 or pap1-1) requires the
function of nuclear exosome components (Rrp6p, Rrp4p, and Mtr4p) (Hilleren et
al., 2001; Libri et al., 2002). These authors envisage the exosome as a
gatekeeper for mRNA export, responsible for retaining and degrading improperly
cleaved or hyperadenylated RNAs in the nucleus (see Figure 4). The exosome
consists of at least ten different proteins with 3’ to 5’ exonuclease activity
(Allmang et al., 1999b; Mitchell et al., 1997) and was first described as a
conserved protein complex required for processing 3’ ends of several small

Figure 4. The exosome functions to retain improperly processed mRNAs in the nucleus. Rrp6p and several components
of the exosome are required for retention of hyperadenylated and hypoadenylated RNAs in the nucleus of
mRNA export and 3’ end formation mutants. See text for details.

TREX complex
Retention and degradation
of hyperadenylated RNAs
r\
r\

42
nuclear RNAs including the 5.8S hbosomal RNA (rRNA), small nucleolar RNAs
(snoRNAs) and U4 snRNA (Allmang et al., 1999a; Mitchell et al., 1997). More
recently, it has been demonstrated that normal mRNAs are also degraded in the
nucleus by the exosome (Das et al., 2003; Jacobs Anderson and Parker, 1998).
Deletion of nup116 leads to the retention and RRP6-dependent degradation of
normal poly(A)+ RNA in the nucleus (Das et al., 2003). In addition to Rrp6p, the
yCBC and a 5' to 3’ nuclear exonuclease (Ratlp) are required for degradation of
nuclear poly(A)+ RNA in the nup116A strain. These authors propose that a
nuclear mRNA degradation (DRN) system, consisting of the CBC and nuclear
exosome, is responsible for general mRNA turnover.
How are polyadenylated mRNAs released from the site of transcription
and exported to the cytoplasm? Is there an export adapter for the poly(A) tail,
analogous to Yralp, that actively recruits an export receptor to the mRNA? Does
a protein bind the poly(A) tail and inhibit CBC-Rrp6p recruitment to the exosome
for retention and degradation in the nucleus? Answers to these questions may
provide a more complete understanding of the mechanisms regulating
polyadenylation, transcript release and mRNA export.

MATERIALS AND METHODS
Yeast and Bacterial Culture Media
Yeast cells were cultured at specified temperatures in YPD media
consisting of 1% Bacto-yeast extract (Fisher), 2% Bactopeptone (Fisher), 2% of
dextrose (Fisher), and 2% Difco-Agar (for plates), unless otherwise indicated.
For growth of the nat>2ztpGAL:NAB2 strain, 2% galactose (Sigma) was
substituted for dextrose. Gene expression from plasmids was achieved by
growing yeast cells in synthetic media (.67% Bacto-yeast nitrogen base without
amino acids [Difco], supplemented with the appropriate L-amino acids [Sigma] as
needed) with dextrose (SD) or galactose (SG) as previously described (Rose et
al., 1990). The addition of 5-flouro-orotic acid (5-FOA) (Diagnostic Chem. Ltd.) to
SD media allowed for the eviction of URA3 marked plasmids when necessary
(Sikorski and Boeke, 1991). Diploid cells were induced to sporulate by growth
overnight on Pre-Sporulation (Pre-Spo) plates (0.8% Bacto-yeast extract, 0.003%
Bacto-peptone, 10% dextrose) followed by 2-3 days of growth on sporulation
(SpoX) plates (1% KOCH3, 0.025 mg/ml ZnOCH3).
Plasmid propagation in bacteria was carried out by growth at 37° C in
Luria-Bertani media (1% Bacto-tryptone, 0.5% Bacto-yeast extract, 1% NaCI, pH
7.5, and 2% Difco-agar for plates) supplemented with 100 \ig/m\ ampicilin
(Sigma) or 40 pg/ml kanamycin (Roche) as required.
43

44
Yeast Strains and Plasmids
The yeast strains and plasmids described in this study are listed in the
appendix. For preparation of full-length recombinant Nab2p, primer set
MSS1131/MSS1132 was designed to amplify the NAB2 gene from L4717
genomic DNA with sequence encoding six histidine residues (Hiss) fused to the
3’ end of the open reading frame. The PCR product was cloned into pGEX-4T-1
(Amersham Pharmaciea Biotech) between the EcoRI and Sa/I sites. The
resulting pGST-Nab2-His6 plasmid was transformed into BL21-CodonPlus (DE3)
cells (Stratagene). The expressed GST-Nab2-His6 protein was purified using
standard gluatathione-Sepharose (Amersham Pharacia Biotech) and His-Bind
(Novagen) chromatographic techniques.
The na£>2z\pPAB1NL strain was constructed by amplification of the PAB1
open reading frame from L4717 genomic DNA using MSS1261 and MSS1259
followed by insertion into Xho\-BstE\\-cui pYES2/SPB4 (Invitrogen) to generate
an NLS-PAB1-V5 gene fusion (pPAB1.3). NLS-PAB1-V5 was excised from
pPAB1.3 by Smal-Xbal digestion and inserted into pRS315 (LEU2, CEN,
[(Sikorski and Hieter, 1989)]) to generate pPAB1.4. The PAB1 5’ and 3’
untranslated regions (UTRs) were amplified by PCR from L4717 genomic DNA
using MSS1287/MSS1263 and MSS1268/MSS1288 primer pairs, respectively.
The 3’ UTR PCR fragment was inserted between Xbal and Sacl sites and the 5’
UTR between Sal I and Sma\ sites of pPAB1.4 to generate the pPAB1.6 plasmid.
The mutant NLS (mnl) sequence was fused to the 5’ of the PAB1 ORF by PCR
amplication from L4717 genomic DNA using primers MSS1262 and MSS1259.

45
The PAB1mni DNA fragment was inserted into Smal-Sphl-cut pPAB1.6, resulting
in pPAB1.7. The pPAB1.6 and pPAB1.7 plasmids were transformed into the
na£>24pGAL::NAB2 shuttle strain (YRH201c) to create the na¿»2ápPAB1NL
(YKN105) and na£>24pPAB1mni (YKN106) strains, respectively. Transformed
cells were initially plated on SG media without leucine (SG-Leu) and then on
YPD to test for nab2A suppression. For YKN105, cells were also streaked to 5-
FOA plates to select for the loss of pGAL::NAB2 (URA3, CEN, (Anderson,
1995)).
The NAB2 allele for tandem affinity purification (NAB2T) was constructed
by amplification of the calmodulin binding peptide (CBP), tabbaco etch virus
(TEV) protease site, protein A (ProtA) sequences from pBS1479 (Rigaut et al.,
1999) using primers MSS811 and MSS812. The resulting PCR fragment also
contained 50 nucleotides of homology to the 3’ end of the NAB2 open reading
frame fused in-frame to the TAP tag followed by sequence encoding the TRP1
gene and another 50 nucleotides of homology to the 3’ UTR of NAB2. After
transformation of the DNA fragment into L4717 cells, tryptophan auxotrophs were
selected and expression of the Nab2pT fusion protein (YKN206) was confirmed
by immunoblot analysis using aNab2 3F2 (1:1000).
For tandem affinity purification of the Nab2NpT and nab2-20NpT peptides,
the 5’ and 3’ UTRs of NAB2 were amplified from L4717 genomic DNA with primer
sets MSS1356/MSS1573 and MSS1384/MSS1358, respectively. Insertion of the
5’ UTR fragment into Sa/l-Psfl-cut and the 3’ UTR fragment into BamFH-Sacl-cut
pRS315 yielded plasmid pNAB2.46. The TAP tag was excised from pBS1479

46
(Rigaut et al., 1999) using BamHI and Hind III restriction enzymes and inserted
into pNAB2.46 to generate pNAB2.47. Primers with sequence encoding the
SV40 NLS and c-Myc epitope in-frame with the open reading frame of NAB2
(MSS1574 and MSS1575) were created and used to amplify the wild-type NES
of NAB2 from L4717 genomic DNA and the mutant NES of nab2-20 from pnab2-
20. The pNAB2.47 plasmid was digested with Pst\ and BamHI and the wild-type
NES (pNAB2.48) and mutant NES (pNAB2.54) fragments were inserted into the
digested plasmid. The NAB2NT (YKN284) and nab2-20NT (YKN287) strains
were created by transformation of pNAB2.48 and pNAB2.54 into L4717,
respectively. An immunoblot analysis of the YKN284 and YKN287 strains was
performed using aNab2 3F2 to confirm expression of the tagged peptides in vivo
(data not shown).
Strains expressing the AD-NT1 and AD-NT2 peptides for epitope mapping
experiments were prepared by transforming pNAB2.42 and pNAB2.41 into
AH109, respectively. The first N-terminal truncation of NAB2 (NT1) was amplified
from L4717 genomic DNA using primer set MSS1423/MSS1444 and the second
N-terminal trunction (NT2) was amplified from L4717 genomic DNA using primers
MSS1423 and MSS1450. Both fragments were inserted into EcoRI-BamHI-cut
pGADT7 (Clontech) to give plasmids pNAB2.42 (NT1) and pNAB2.41 (NT2).
The in vitro glutathione S-transferase pull-down experiments were
performed by creating plasmids containing GST-LRR (pMEX67.5), GST-SUB2
(pSUB2.5), GST-PAB1 (pPAB1.8), GST-YRA1 (pYRA1.17), and GST-NPL3
(pGST-NAB1) gene fusions followed by transformation into BL21-CodonPlus

47
(DE3) cells for inducible expression of the fusion proteins. Briefly, the LRR of
MEX67 was amplified from L4717 genomic DNA with primers MSS1661 and
MSS1662 and inserted into EcoRI-Sa/l-cut pGEX-4T-1. The open reading
frames of SUB2 and PAB1 were amplified from L4717 genomic DNA using
primer sets MSS1729/MSS1730 and MSS1727/MSS1728, respectively. SUB2
and PAB1 were inserted into pGEX-4T-1 after digestion with BamHI and Xho\.
Since the YRA1 gene contains a single intron, E1 was amplified from L4717
genomic DNA using primer set MSS1539/MSS1540 and E2 with primers
MSS1541 and MSS1542. The E1 PCR fragment was inserted into BamHI-
EcoRI-digested pGEX-4T-1 to give pYRA1.16 and E2 was inserted into EcoRI-
X/iol-cut pYRA1.16, creating pYRA1.17. The NAB2 N-terminal fragment was
excised from pNAB2.41 by digestion with A/col and BamHI and inserted into
pET15b (Novagen) to create pNAB2.58. The N-terminal region of nab2-20 was
amplified from pnab2-20 (Anderson, 1995) with MSS1423 and MSS1444 and
inserted into EcoRI-BamHI-cut pGADT7 to create pNAB2.45. The mutant N-
terminal fragment was excised from pNAB2.45 by digestion with A/col and BamHI
and inserted into pET15b to create pNAB2.59. The pNAB2.58 and pNAB2.59
plasmids were transformed into BL21-CodonPlus (DE3) cells for inducible
expression of the Nab2pN and Nab2-20pN fragments, respectively.
Nucleic Acid Isolation Procedures
Yeast genomic DNA was isolated from 5 ml of logarithmically growing
(OD6oo = 0.5-2 U/ml) L417 cells. Cells were pelleted by centrifugation at 1,500
x g for 5 minutes. The pellet was re-suspended in 0.5 ml of cell wall digestion

48
buffer (800 mM Sorbitol, 100 mM EDTA, 30 mM DTT) and 50 pi of 5 mg/ml
Zymolyase 100T (Seikagaku Corporation). Cell walls were digested for 1 hour at
37° C and spheroplasts were harvested by centrifugation at 3,000 x g for 1
minute. Spheroplasts were re-suspended in 0.5 ml of lysis buffer (50 mM Tris,
20 mM EDTA, 1% SDS) and incubated at 65° C for 30 minutes with mixing every
10 minutes. Proteins and cell debris were precipitated by incubating on ice in the
presence of 1.8 M KOAC and pelleted at 4° C by centrifugation at 14,000 x g for
15 minutes. The supernatant was transferred to a fresh tube and DNA was
precipitated in 50% isopropanol for 10 minutes at 24° C. The DNA was pelleted
at 14,000 x g for 1 minute, washed and dried in a SpeedVac (Savant). After re¬
suspension of the pellet in 250 pi of TE buffer (10 mM Tris-HCI pH 7.5, 1 mM
EDTA), RNA was digested at 37° C for 1 hour using 200 ng of RNase A.
Proteins were extracted with phenol and genomic DNA was precipitated using
0.3 M sodium acetate. DNA was recovered by centrifugation at 14,000 x g,
washed, dried and resuspended in 100 pi of TE. The concentration of genomic
DNA was determined by measuring the absorbance of 260 nm light (OD26o)-
Previously described methods were used to recover plasmids from yeast cells
(Strathern and Higgins, 1991) and bacterial cells (Sambrook etal., 1989).
Total RNA was extracted from yeast cells using 65° C acid equilibrated
phenol as previously described (Guthrie and Fink, 1991). Briefly, logarithmically
growing cells were harvested by centrifugation at 2,000 x g for 5 minutes and re¬
suspended in AE lysis buffer (50 mM NaOC3H, pH 5.3, 10 mM EDTA, 1% SDS).
Proteins and cell debris were extracted from the supernatant at 65° C with AE

49
equilibrated phenol. RNA was precipitated at -70° C for 1 hour using 2 M
ammonium acetate. After centrifugation, pelleted RNA was re-suspended in
diethyl pyrocarbonate (DEPC) treated d2H20 and the concentration was
determined by measuring the OD26o.
Cell Transformation
Transformation of yeast cells was performed by treatment with lithium
acetate (Ito et al., 1983). Cells were grown in YPD or SD to an OD6oo = 0.5 U/ml.
A total of 10 ml for each transformation was harvested by centrifugation at 1,500
x g for 5 minutes. Cells were washed with d2H20 and harvested again by
centrifugation. The cell pellet was re-suspended in 700 pi of buffer (Tris-EDTA,
0.1 M lithium acetate, 43% polyethylene glycol molecular weight 3350) containing
5 pg of denatured calf thymus DNA and 1-5 pg of plasmid DNA. Incubation was
carried out at 24° C for 30 minutes, rotating end over end. At last, cells were
heat-shocked at 42° C for 15 minutes, washed, and plated on SD plates
supplemented with the appropriate amino acids.
Electro-competent bacterial cells (DH5a) were transformed with plasmid
DNA (1-100 ng) using an electric current generated by a BioRad GenePulser
(2.5 kV, 200 Q, 25 pFd). After electro-poration and DNA uptake, cells were
allowed to recover in LB media at 37° C for 1 hour. The cell suspension was
plated on LB supplemented with appropriate antibiotics and incubated overnight
at 37° C to allow for colony formation.

50
Yeast Genetic Manipulations
Mating, sporulation, and tetrad dissections were all performed as
described (Guthrie and Fink, 1991). Haploid cells of complementing mating type
(mat a and mat a) strains were patched on YPD plates at 24° C for 1-2 days.
Diploids were selected on SD media containing the appropriate amino acids.
Spore formation was achieved by incubating diploids on Pre-Spo plates for one
day and SpoX plates for two days. Cell walls were digested for 10 minutes in 1.0
M sorbitol and 0.5 mg/ml Zymolyase 20T (Seikagaku Corporation) releasing
individual spores for micromanipulation. Dissected spores were allowed to
germinate and form colonies on YPD plates at 24° C unless otherwise indicated.
Colonies were tested for various auxotrophic markers and growth at specified
temperatures. The inability of individual spores to form colonies was also noted.
Yeast Total Cell Protein Isolation
Five OD6oo units of yeast cells were harvested by centrifugation at 2,000 x
g. Cells were washed once with d2H20 and re-suspended in 200 pi of 10%
trichloroacetic acid (TCA) (Fisher) in a microfuge tube. Acid-washed beads were
added to the cell suspension. The tubes were vortexed vigorously for 10
seconds and chilled on ice for 15 seconds. Vortexing was repeated for a total of
six times. The lysate was removed from the beads and subjected to
centrifugation at 14,000 x g for 10 minutes. The protein pellet was recovered
after centrifugation and re-suspended in 100 pi of Laemmli cocktail (10 mM Tris-
HCI pH 6.8, 4% SDS, 20% glycerol, 1.3 M (3-mercaptoethanol, 4 mg/ml
Bromphenol blue). The protein cocktail was neutralized with 1 M Tris-base and

51
boiled for 3 minutes prior to fractionation on 12.5% polyacrylamide-1 % SDS gels,
unless otherwise noted.
Fluorescence In Situ Hybridization and Cellular Immunofluorescence
In order to determine the subcellular localization of proteins and poly(A)+
RNA, yeast cells were grown to an OD6oo = 0.1 - 0.5 U/ml. After harvesting by
centrifugtion at 1,000 x g for 10 minutes, cells were fixed in freshly prepared 4%
formaldehyde (from EM grade paraformaldehyde) for 2 hours with gentle rocking.
Fixed cells were pelleted at 800 x g for 2 minutes and washed twice with WB1
(100 mM KH2P04, pH 6.5) and once with WB2 (100 mM KH2P04, pH 6.5, 1.2 M
Sorbitol). Cells were harvested as before and re-suspended in 1 ml of WB2 plus
30 mM p-mercaptoethanol. Spheroplast formation was achieved by the addition
of 300 ng of Zymolyase 100T and incubation at room temperature for 30-45
minutes. After greater than 90% of the cells no longer contained cell walls as
determined by phase-contrast microscopy (400X magnification), cells were
collected by centrifugation at 2000 x g for 1 minute. Cells were washed with
WB2 and attached to polylysine-coated 10-well HTC Blue Slides (Cell-Line
Associated). Slides were washed once in ice-cold PBS (150 mM NaCI, 3 mM
KCI, 10 mM Na2HP04, 2 mM KH2P04, pH 7.4) and dipped in 100% methanol for
5 minutes at -20° C followed by 100% acetone for 30 seconds. After three
washes with PBS, slides were placed in PBS plus 0.1% Triton-X (Sigma) for 5
minutes and washed three more times with PBS. Slide wells were blocked for
non-specific antibody interactions by the addition of 3 % BSA/PBS for 30 minutes
at 24° C. Primary antibodies were diluted in 3% BSA/PBS (aNab2, 1:500;

52
aPabl, 1:5000; aNop1, 1:500; aV5, 1:500; aDig, 1:100) and added to respective
wells for a 60 minute incubation at 24° C. Poly(A)+ RNA was detected by
fluorescence in situ hybridization (FISH) using a d(T)5o-digoxigenin conjugated
probe according to a previously published procedure (Amberg et al., 1992). The
Alexa Fluor 488 goat anti-mouse IgGi secondary antibody was used to detect
aNab2, aPabl, aV5 and aDig. For co-localization of poly(A)+ RNA and Nopip,
the anti-Nop1 A66 antibody (gift from John Aris, University of Florida, Gainesville,
FL) was detected by the Alexa Fluor 555 goat anti-mouse lgG2A secondary
antibody (Molecular Probes). DNA was detected by 0.5 pg/ml 4’6-diamidino-2-
phenylindole (DAPI). Cells were visualized using an Axiophot (Zeiss)
microscope equipped with a X100 fluorescence/differential contrast objective and
images were captured by either monochrome (Zeiss) or color (Spot) digital
cameras.
In Vitro 3’ End Processing Assays
Standard 3’-end processing assays were performed essentially as
described (Minvielle-Sebastia et al., 1997) with 2 pi of either YAS394 (pab1A) or
BMA64 (PAB1) cell extracts. Reconstitution assays with purified factors were
performed with 0.5 pi of partially purified CF I (Mono Q fraction from CFII
purification; (Minvielle-Sebastia et al., 1998)) and 0.5 pi of CPF (affinity purified
from strain YSD10 expressing a TAP-tagged Fiplp fusion protein; unpublished
data). Reactions were supplemented with either 25-800 ng of GST-Nab2p-His6,
50-800 ng of GST-Nab4p (Minvielle-Sebastia et al., 1998), or 50-1000 ng of
purified bovine PABPN1 (gift of Elmar Wahle, University of Halle-Wittenberg,

53
Halle Saale, Germany). The polyadenylation time course experiment was
performed with a 12-fold reaction mixture of YAS394 extract supplemented with
9.6 pg of GST-Nab2p-His6 and incubated at 30° C for 15 minutes without RNA
substrate. Following addition of labeled transcript, aliquots of 20 pi were
withdrawn at the specified times. Reactions were analyzed on 40 cm long 6%
polyacrylamide-8.3 M urea gels.
Filter Binding Assays
Standard filter binding assays with r(A)25 (Dharmacon Research) and
either GST-Nab2p-His6 or GST-His6 were performed as previously described
(Wong and Lohman, 1993). Binding reactions were incubated at 30° C for 30
minutes in 20 mM Tris-HCI pH 7.4, 100 mM KCI, 2.5 mM MgCI2, 0.5 U/pl
Superase-ln (Ambion) with 5 pM 32P end-labeled A25 RNA and increasing
amounts of purified GST-Nab2p-His6 or GST-HÍS6 fusion proteins. Protein-RNA
complexes were filtered on 0.45 micron nitrocellulose (Osmonics) and bound
versus unbound RNA fractions were quantified using a Storm phosphorlmager
(Molecular Dynamics). Each point on the curve represents the mean of three
independent experiments.
Tandem Affinity Purification
Total protein extracts of YKN206 were prepared by growing cells at 30° C in
2.5 L of YPD medium to an OD6oo = 2-4 U/ml. Cells were harvested by
centrifugation and resuspended in 1 volume of Buffer A(TAP) (10 mM K-HEPES,
pH 7.9, 10 mM KCI, 1.5 mM MgCI2, .5 mM DTT) with protease inhibitors (.5 mM
PMSF, 2 mM Benzamidine, 1 pM leupeptin, 2 pM pepstatin, 4 pM chymostatin,

54
2.6 pM aprotinin). Cells were lysed by two subsequent passages through a
French-press at 1,200 PSI. After lysis, KCI was added to a final concentration of
200 mM and the supernatant was clarified by centrifugation at 60,000 x g for 30
minutes and 130,000 x g for 90 minutes. The supernatant was dialyzed against
1000X volume of Buffer D(TAP) (20 mM K-HEPES, pH 7.9, 50 mM KCI, .2 mM
EDTA, .5 mM DTT, 20% v/v glycerol) with protease inhibitors (.5 mM PMSF, 2
mM Benzamidine). Protein extracts were added to 200 pi of IgG-Sepharose
beads (Amersham) and incubated at 4° C for 2 hours. The beads were washed
three times with IPP150 Buffer (10 mM Tris-CI, pH 8.0, 150 mM NaCI, .1% NP40)
and then incubated with 1 ml of TEV Cleavage Buffer (10 mM Tris-CI, pH 8.0,
150 mM NaCI, .5 mM EDTA, .1% NP40, 1 mM DTT) and 100 units of TEV
protease at 16° C for 2 hours. Three volumes of calmodulin binding buffer (10
mM Tris-CI, pH 8.0, 150 mM NaCI, 1 mM MgOAC, 1 mM imidazole, 2 mM CaCh,
.1% NP40, 10 mM (3-mercaptoethanol) and 200 pi of calmodulin affinity resin
(Stratagene) was added to the TEV eluate and incubated at 4° C for 1 hour.
After three washes with calmodulin binding buffer, Nab2pCBP was eluted using
800 pi of calmodulin elution buffer (10 mM Tris-CI, pH 8.0, 150 mM NaCI, 1 mM
MgOAC, 1 mM imidazole, 2 mM EGTA, .1% NP40, 10 mM p-mercaptoethanol).
For protein analysis, 400 pi of the calmodulin eluate was precipitated using 10%
TCA, pelleted at 14,000 x g, and re-suspendend in 10 pi of Laemmli cocktail.
The protein cocktail was neutralized with 1 M Tris-base and boiled for 3 minutes
prior to fractionation on a 12.5% polyacrylamide-1 % SDS gel. Proteins were
visualized using either Coomassie blue stain (2.5 mg/L Coomassie blue dye

55
[BioRad], 50% methanol, 10% acetic acid) or silver stain (0.1% Silver Nitrate).
Proteins were identified by either matrix assisted laser desorption time-of-flight
mass spectrometry (MALDI-TOF MS) in collaboration with John Aitchison and
David Dilworth (Institute for Systems Biology, Seattle, WA) or immunoblot
analysis as previously described (Anderson et al., 1993b) using the following
antibodies and dilutions: aNab2 3F2 (1:1000), aNab2 1F2 (1:500), aKap104
1D12 (1:1000), aPabl 1G1 (1:1000), «Nab3 2F12 (1:250), cxNab4 3H1 (1:500),
aNpl3 (1:500) (gift from Pamela Silver, Harvard Medical School, Boston, MA),
aMex67 (1:500) (gift from Ed Hurt, University of Heidelberg, Heidelberg,
Germany), aYral (1:1000) (gift from Douglas Kellogg, University of California,
Santa Cruz, CA) and horseradish peroxidase-conjugated sheep anti-mouse
secondary (1:5000) (Amersham Pharmacia Biotech).
Preparation of Polyclonal Antisera and Monoclonal Antibodies
The University of Florida Interdisciplinary Center for Biotechnology Research
(ICBR) hybridoma laboratory was responsible for preparation of the Nab2pTAP
polyclonal antiserum and aKap104 1D12 monoclonal antibody. Two injections (5
ptg) of purified Nab2pTAP were performed on BALB/c mice (Harlow and Lane,
1988). Ten days after the second injection, a preliminary bleed was performed
and dilutions (1:100, 1:250, 1:500) of the blood were tested for immunoreactivity
against Nab2pT-associated proteins. The polyclonal antiserum (1:500) was used
to screen a X.gt 11 expression library for recombinant yeast genes as previously
described (Snyder et al., 1987). Positive plaques were purified, cloned and

56
sequenced. Identification of genes was accomplished by searching the
Saccharomyces Genome Database (SGD) for matching DNA sequences.
To isolate the aKap104 monoclonal antibody, spleen cells of the mouse were
fused with SP2/0 myeloma cells as previously described (Harlow and Lane,
1988). The 1D12 monoclonal antibody reacted against A.gt 11 phage expressing
KAP104.
Polv(A) Tail Length Determination
Total RNA was prepared from whole cells and poly(A) tail lengths were
determined as previously described (Minvielle-Sebastia et al., 1998). Briefly,
total RNA (1 pg) was 3’-end labeled at 0° C for 20 hours with [32P]pCp (NEN)
using 20 U of T4 RNA ligase (New England Biolabs) in a 30 pi reaction mix (50
mM HEPES pH 8.3, 5 pM ATP, 10 mM MgCI2, 3.3 mM DTT, 10% DMSO, 300
pg/ml acetylated BSA). Non-poly(A) RNA was digested at 37° C for 2 hours
using RNases A (50 ng/pl) and T1 (1 U/pl) in 10 mM Tris-HCI pH 7.5 and 300
mM NaCI. The digestions were stopped by the addition of EDTA, proteins were
extracted with phenol and RNA was precipitated using 2 M ammonium acetate
and 5 pg of RNase-free glycogen (Boehringer Mannheim). Poly(A) tails were
fractionated on 8% polyacrylamide-8.3 M urea gels and visualized by
autoradiography.
In Vitro GST Pull-down Experiments
Direct interactions between the N-terminus of Nab2p and either the LRR
of Mex67p, Pablp or Sub2p were demonstrated using the ProFoundâ„¢ Pull-Down
GST Protein:Protein Interaction Kit (Pierce Biotechnology). Induction of

57
recombinant proteins in strains carrying pNAB2.58 (EKN102), pNAB2.59
(EKN103), pMEX67.4 (EKN104), pSUB2.4 (EKN111), pPAB1.8 (EKN110),
pGST-NAB1 (EKN109), pYRA1,17 (EKN114) or pGEX-4T-1 (EKN101) plasmids
was achieved by adding 1 mM IPTG (Sigma) to TB+K (1.2% Bacto-tryptone,
2.4% Yeast Extract, 17 mM KH2P04, 85 mM K2PO4, 0.4% glycerol, pH 7.0)
growth media and growing cells at 37° C for 2 hours. Cells were pelleted and re¬
suspended in 200 pi of TBS (25 mM Tris-HCI pH 7.2, 150 mM NaCI) with
protease inhibitors (5 pg/ml pepstatin A, 1 pg/ml chymostatin, 1 mM e-
aminocaproic acid, 1 mM p-aminobenzamidine, 1 pg/ml leupeptin, 2 pg/ml
aprotinin). Cells were lysed by adding 200 pi of ProFoundâ„¢ Lysis Buffer (Pierce
Biotechnology) and the supernatant was clarified by centrifugation at 14,000 x g
for 5 minutes. GST fusion proteins were bound to glutathione-Sepharose beads
(Amersham Pharmacia Biotech) by incubating 800 pi of supernatant with 30 pi of
beads at 4° C for 2 hours. After three washes with 800 pi of wash buffer (50%
ProFoundâ„¢ Lysis Buffer, 12.5 mM Tris-HCI pH 7.2, 75 mM NaCI), an equal
volume of either EKN102 or EKN103 cell lysate was added to GST pre-bound
Sepharose beads. Binding reactions were carried out at 4° C for 3 hours. Beads
were washed three times with wash buffer and proteins were eluted by boiling at
100° C for 3 minutes in 20 pi of Leammli cocktail. One-half of each binding
reaction was loaded onto a 12.5% polyacrylamide-1 % SDS gel and proteins were
detected using either aNab2 1F2 or aGST (gift from the University of Florida
ICBR hybridoma core).

58
Phosphatase Treatment with k Phosphatase
For phosphatase treatment of Nab2p in mRNA export mutant strains, cells
(5 OD6oo units) were lysed by glass bead vortexing in 250 pi of lysis buffer (50
mM Tris-HCI pH 7.5, 100 mM NaCI, 10 mM imidazole, 1 mM EDTA, 0.2 mM
Na3V04, 10 mM NaF, 0.5 mM PMSF, 5 pg/ml pepstatin A, 1 pg/ml chymostatin, 1
mM e-aminocaproic acid, 1 mM p-aminobenzamidine, 1 pg/ml leupeptin, 2 pg/ml
aprotinin). De-phosphorylation was performed by adding 400 units of k
phosphatase (New England Bio-labs) to 5 pi of lysate according to the
manufacturer’s instructions. Reactions were stopped by precipitation of proteins
in 10% TCA. Proteins were re-suspended in Laemmli cocktail and fractionated
on 8% polyacrylamide-1 % SDS gels. The Nab2p isoforms were detected by
immunoblot analysis using aNab2 3F2. UV-light induced photocrosslinking was
performed as described (Wilson et al., 1994).

RESULTS
Research Objectives
Efficient and reliable gene expression in eukaryotic cells requires precise
temporal and spatial coordination of many nuclear and cytoplasmic events.
Nuclear events include mRNA transcription, capping, splicing, polyadenylation,
and nuclear export whereas protein translation, and mRNA turnover occur in the
cytoplasm. RNAP II coordinates nuclear processing events with transcription by
recruiting the appropriate protein complexes to the 5’ ends, introns and 3’ ends of
pre-mRNA (reviewed in Bentley, 2002; Hirose and Manley, 2000; Proudfoot et
al., 2002; Steinmetz, 1997). In addition, proteins that bind to the 7-
methylguanylate cap stimulate splicing, polyadenylation and nuclear export of
mRNA (Lewis and Izaurralde, 1997; Shatkin and Manley, 2000). Factors
required for efficient splicing, in turn, interact with proteins required for
polyadenylation and mRNA export (Lei and Silver, 2002; Luo et al., 2001; Lutz et
al., 1996; Strasser and Hurt, 2001; Takagaki et al., 1996; Vagner et al., 2000)
providing further evidence that nuclear processing events, from transcription to
nuclear export, are intimately linked.
During transcription an abundant class of heterogeneous RNA binding
proteins (hnRNPs) also associate with pre-mRNA and are believed to play a role
in the coordination of pre-mRNA processing events and nuclear mRNA export by
59

60
binding to specific RNA sequences and serving as export adaptors for
karyopherins (Dreyfuss et al., 2002; Dreyfuss et al., 1993; Krecic and Swanson,
1999; Lei and Silver, 2002; Nakielny and Dreyfuss, 1999). This view of hnRNPs
as active participants in pre-mRNA processing, particularly mRNA export, is
controversial. Reed and Magni (2002) have proposed that hnRNPs bind
primarily to introns and need to be removed from the mRNA-Particle (mRNP)
before export while splicing-dependent SR proteins and conserved mRNA export
factors (Yralp and REF/Aly) actively recruit nuclear export receptors (Mex67p
and TAP) to the mRNP. An alternative possibility is that hnRNPs act at the
interface between nuclear RNA processing events and mRNA export to precisely
orchestrate the dissociation of the RNA processing machinery prior to the
recruitment of export receptors. The goal of my thesis research has been to test
the latter hypothesis using the yeast hnRNP Nab2p.
Nab2p was first identified as a nuclear polyadenylated RNA binding
protein required for vegetative growth in yeast (Anderson et al., 1993b). In
addition to loss of viability, depletion of Nab2p resulted in an accumulation of
hyperadenylated RNA in the nucleus (Hector, 2000; Hector et al., 2002). These
phenotypes mirror those for several mRNA export mutants, including prp20,
mtr2, rat7, rat8, mex67, gle1, and rip1 (Forrester et al., 1992; Hilleren and
Parker, 2001; Jensen et al., 2001b; Kadowaki et al., 1994; Tseng et al., 1998).
Unique to NAB2, however, a mutant was isolated that uncouples the
polyadenylation defect from the export block. At temperatures permissive for
growth, nab2-21 cells exhibit hyperadenylated tails but do not accumulate

61
poly(A)+ RNA in the nucleus, whereas at temperatures restrictive for growth,
poly(A)+ RNA export is also blocked. Interestingly, PAB1 was isolated as a high
copy suppressor of nab2A growth defects but increased expression of Pablp did
not resolve the hyperadenylation defect and only mildly restored poly(A)+ RNA
export to wild-type levels (Hector, 2000; Hector et al., 2002). Altogether, these
results suggest Nab2p performs dual functions in vivo. First, Nab2p terminates
polyadenylation, possibly analogous to PABPN1, by directly binding to the
polyadenylate tail. Secondly, after binding and restricting tail length, Nab2p
coordinates the release from RNAP II and serves as an adapter for the nuclear
export of poly(A)+ RNA. The following specific aims were designed to provide
evidence for this model: (1) demonstrate Nab2p, like PABPN1, binds to poly(A)+
RNA homopolymers and limits poly(A)+ tail length in vitro; (2) determine if NAB2,
but not PAB1, is responsible for limiting poly(A)+ tail lengths and promoting
mRNA export in vivo; (3) identify the factors, in combination with Nab2p, involved
in the nuclear export of polyadenylated mRNA.
Nab2p Binds Polv(A)+ RNA and Limits Polv(A) Tail Length In Vitro
The maturation of eukaryotic mRNA 3’ ends first requires cleavage at
specific sequences upstream of the transcriptional termination site by a group of
cleavage factors, followed immediately by the addition of adenosine residues to
the cleaved pre-mRNA (for reviews see Colgan and Manley, 1997; Proudfoot and
O'Sullivan, 2002; Wahle and Keller, 1996; Zhao et al., 1999). While the number
of adenosines added to a given mRNA can vary slightly depending on the cell
type, developmental stage and growth conditions, poly(A) tails are generally

62
found to be between 200-300 nucleotides in mammals and 70-90 nucleotides in
Saccharomyces cerevisiae (Wahle and Ruegsegger, 1999). When cells are
depleted of Nab2p, however, poly(A) tail lengths increase dramatically up to 600
nucleotides (Hector et al., 2002).
Expression and Purification of Recombinant GST-Nab2p-Hisfi Protein
In order to determine if aberrant tail length regulation observed in nab2A
cells is directly related to Nab2p function, a recombinant Nab2p (rNab2p) was
produced and used for in vitro studies. Briefly, DNA sequence encoding a
glutathione S-transferase (GST) protein was cloned 5’ of the NAB2 start codon to
generate a GST-Nab2p fusion product. The GST-Nab2p was expressed in E.
coli cells and purified using a glutathione column. Unfortunately, a majority of the
proteins eluted from the glutathione column were breakdown products of Nab2p
(Figure 5A, GST-Nab2p lane). In an attempt to purify full-length rNab2p away
from the degradation products, six histidine (Hise) residues were fused to the
carboxyl terminus of GST-Nab2p to allow for an additional purification step using
a nickel column. The use of both tags for purification purposes greatly increased
the amount of full-length rNab2p that was recovered (Figure 5A, GST-Nab2p-His6
lane).
rNab2p Binds with High Affinity to Polv(A) RNA Homopolymers
The affinity of Nab2p for poly(A) RNA was determined using a standard
filter-binding assay and (rA)25 (Wong and Lohman, 1993). As shown in Figure
5B, the observed dissociation constant (Kd) was 10.5 nM (blue squares).
Importantly, GST-His6 protein had little or no affinity for poly(A)+ RNA (Figure 5B,

Figure 5. Nab2p binds strongly to poly(A)+ RNA homopolymers. A) Purification using both GST and His6 binding columns
results in greater full-length product (GST-Nab2p-His6 lane) than GST alone (GST-Nab2p lane). Molecular
weight standards are indicated. B) A standard filter-binding assay with (rA)25 and recombinant GST-Nab2p-His6
revealed a high affinity of Nab2p for poly(A)+ RNA (blue squares). GST-His6 protein had very little affinity for
(rA)25 (green circles).

TT"rrl—' ' — —
100 10 s 10'7 10e
[protein, M]
100
80
60
40
20
0
CT>

65
green circles) and bovine poly(A)-binding protein (PABPN1) has been shown
previously to have an affinity for (rA)25 (K¿ = 7 nA/f; Gorlach et al., 1994)
comparable to Nab2p. Considering Nab2p binds strongly to poly(A)+ RNA and is
localized to the nucleus of yeast cells (Anderson et al., 1993b), we reasoned that
Nab2p functions to restrict poly(A) tail lengths. In vitro assays were utilized to
assess the role of Nab2p in restricting poly(A) tail length during polyadenylation.
Nab2p Restricts Polv(A) Tail Length In Vitro
Although cleavage and polyadenylation reactions are tightly coupled in
vivo, these two activities can be separated in vitro. A labeled iso-1-cytochrome c
(CYC1) RNA is cleaved and polyadenylated when added to yeast whole cell
protein extracts (WCE) (Butler and Platt, 1988). Remarkably, the addition of
cordycepin triphosphate or substituting CTP for ATP in WCE inhibits
polyadenylation but not cleavage of the precursor CYC1 RNA (Butler et al.,
1990). Alternatively, if the precursor CYC1 RNA is pre-cleaved (CYCIpre) at the
normal cleavage position using a convenient restriction site, this CYCIpre RNA
is still polyadenylated (Lingner et al., 1991a; Lingner et al., 1991b). If pab1A
extracts are used, tail lengths increase to about 200 nucleotides. The addition of
recombinant Pablp to these extracts restricts poly(A) tail lengths to about 60
nucleotides (Amrani et al., 1997; Minvielle-Sebastia et al., 1997). It has been
demonstrated that Pablp binds to poly(A)+ RNA and recruits poly(A) nucleases
(Pan2p and Pan3p) to the tail which degrades the poly(A)+ RNA in a 3’ to 5’
manner (Boeck et al., 1996; Brown et al., 1996). These authors propose poly(A)
tails are normally synthesized very long but Pablp gradually deadenylates tails

66
through the activity of Pan2p and Pan3p (Brown and Sachs, 1998). Maximum
poly(A) tail length (80-100 nucleotides) for pab1 and pan mutants observed in
vivo, however, does not correspond to the maximum tail length exhibited in vitro
(greater than 200 nucleotides) (Brown and Sachs, 1998; Minvielle-Sebastia et al.,
1997) suggesting an additional factor(s) is required for poly(A) tail length
regulation in vivo. To determine if Nab2p can restrict poly(A) tail length in vitro, in
collaboration with Lionel Minvielle-Sebastia (University of Bordeux, France),
increasing amounts of Nab2p was added to YAS394 (pab1A/rrp46A) extracts and
32P-labeled CYCIpre RNA poly(A) tail lengths were examined. Note that in the
presence of Pablp (Figure 6A, PAB1 control lane), poly(A) tail lengths were
restricted to about 60 nucleotides but there was relatively inefficient conversion of
precursor to oligo(A) and from oligo(A) to poly(A) tails. In the pab1A strain,
polyadenylation was unrestricted with very long tails (greater than 200
nucleotides (Figure 6A, pab1A control lane). Importantly, the addition of Nab2p
(Figure 6A, 400-800 ng), restricted tail lengths to approximately 90 nucleotides.
Surprisingly, the addition of low levels of Nab2p stimulated polyadenylation (note
the low levels of precursor and oligo(A) RNA up to 200 ng) (Figure 6A). At 400
ng and greater, Nab2p inhibited polyadenylation. This could be due to the
sequestration of a factor required for efficient polyadenylation in vitro. Indeed,
Nab2p interacts with the CF IB (Nab4p) in the yeast two-hybrid system (Krecic,
1998; Uetz et al., 2000). Nab4p is required for cleavage site selection and
stimulates polyadenylation in combination with CF IA and PAP (Kessler et al.,
1997; Minvielle-Sebastia et al., 1998). As shown in Figure 6B, the addition of

Figure 6. Nab2p is required for restricting poly(A) tail lengths in vitro. A) Nab2p stimulates polyadenylation but inhibits
hyperadenylation in vitro. Reactions were performed using pre-cleaved CYC1 precursor RNA (CYCIpre) and
either YAS394 (pab1A) or wild-type (PAB1) cell extracts. Extracts were either used directly (control lanes) or
supplemented with 100-800 ng of GST-Nab2p-His6. B) Nab4p stimulates hyperadenylation in vitro. Extracts
were supplemented with 50-800 ng of GST-Nab4p. C) PABPN1 stimulates polyadenylation but inhibits
hyperadenylation in vitro. Yeast extracts were supplemented with 50-1000 ng of recombinant bovine PABPN1.
Molecular weights are indicated and polyadenylated RNAs are bracketed (pA). Photos courtesy of L. Minvielle-
Sebastia.

68
<
I I
cn h-
CN CN
cd in
o
N-
i A i i i I
CNOO h- t-OO
O TtCO T- 00)00
CO CNCN CN CNt-t-

69
Nab4p to pab1A extracts stimulates hyperadenylation in vitro suggesting Nab2p
and Nab4p may have antagonistic functions in polyadenylation. Remarkably, the
addition of low levels of bovine PABPN1 (up to 200 ng) restricts tail lengths in the
yeast system while inhibiting polyadenylation at higher concentrations (greater
than 400 ng) (Figure 6C).
These results indicate that Nab2p is necessary for proper tail length
control in vitro using whole-cell extracts, but it was also of interest to determine if
Nab2p is sufficient for tail length restriction in the absence of other cellular
proteins. To accomplish this, rNab2p was added to highly purified cleavage and
polyadenylation factors, CF I and CPF. As before, hyperadenylation of the
CYC1 pre RNA was observed in the absence of Pab1 p (Figure 7A) (Amrani et al.,
1997; Minvielle-Sebastia et al., 1997). The addition of low levels (25-50 ng) of
Nab2p increased polyadenylation efficiency and limited tails to ~90 nucleotides in
length while higher levels of Nab2p (100-200 ng) profoundly inhibited
polyadenylation (Figure 7A). Importantly, this activity of Nab2p was not specific
for the uncoupled polyadenylation reaction because using a non-cleaved CYC1
RNA Nab2p promoted polyadenylation while restricting tail lengths (Figure 7B).
The mechanism for poly(A) restriction by Pablp has been proposed to
involve hyperadenylation followed by deadenylation (Brown and Sachs, 1998)
whereas PABPN1 is believed to bind to the poly(A) tract during synthesis and
terminate polyadenylation at a specific length (Wahle, 1995). To discriminate
between these two potential models for Nab2p activity, the effect of 400 ng of
Nab2p on polyadenylation in pab1A extracts at various time points was

Figure 7. Nab2p inhibits hyperadenylation during synthesis. A) Hyperadenylation is inhibited by the addition of Nab2p to
purified cleavage and polyadenylation factors. Reactions were performed using either purified CF I + CPF or
PAB1 extract (control). Purified factors were supplemented with 10 ng of GST-Nab4p and 25-200 ng of GST-
Nab2p-His6. B) Nab2p inhibits hyperadenylation during coupled cleavag-polyadenylation reactions in vitro.
Extracts were supplemented with 100-800 ng of GST-Nab2p-His6. Uncleaved (CYC1), cleaved (5’), and
polyadenylated RNAs (pA) are indicated. C) Nab2p does not promote tail trimming of hyperadenylated RNA. A
Time course experiment was performed by incubating pab1A extracts with 400 ng of GST-Nab2p-His6 for 2-180
minutes. Photos courtesy of L. Minvielle-Sebastia.

A
CF I + CPF
B
PAB1
pab1A PAB1
L
pab1 A
J

72
determined (Figure 7C). Remarkably, even at the earliest time point (2 min),
Nab2p restricted poly(A) tail length to 90 nucleotides. Over time (up to 180
minutes), no further decrease in poly(A) tail length was observed. These results
are contrary to those obtained with 250 ng of Pablp in which poly(A) tails
successively shorten over time (Minvielle-Sebastia et al., 1997) suggesting the
mechanisms of poly(A) tail length regulation for Nab2p and Pablp are distinct. It
is important to note that wild type extracts (Figure 6A and Figure 6C, PAB1
lanes) exhibit tail lengths about 20 nucleotides shorter than pab1A extracts that
have been augmented with 400 ng of Nab2p. This is likely to reflect the Pab1-
Pan2/3 activity present in wild type extracts, but not in pab1A extracts (Brown
and Sachs, 1998; Brown et al., 1996). We conclude from these results that
Nab2p is an essential nuclear poly(A)-binding protein required for efficient
polyadenylation and poly(A) tail length restriction in vitro.
NAB2 Limits Polv(A) Tail Length and Promotes mRNA Export In Vivo
To explore the possibility that Nab2p is also responsible for poly(A) tail
length restriction in vivo, a high-copy suppressor screen was previously
employed to isolate proteins that could compensate for loss of Nab2p (Hector,
2000). Surprisingly, high-copy PAB1 was the only identified suppressor of the
nab2A growth defect. Initially these results argued that the essential function of
Nab2p is in poly(A) tail length control. Closer inspection of the na£>2/ipPAB1
phenotypes, however, revealed that increased PAB1 expression did not correlate
with significantly shortened poly(A) tails (Hector, 2000; Hector et al., 2002).
Poly(A) tail lengths were approximately 140 nucleotides in length in nab2ApPAB'\

73
cells, a mere 20-30 nucleotides shorter than tail lengths observed in
nai)24pGAL::NAB2 when grown in glucose (Hector et al., 2002). Pablp also
regulates mRNA turnover (Tucker and Parker, 2000; Wilusz et al., 2001) and
promotes translation initiation through interactions with the poly(A) tail and elF4E
(Sachs and Davis, 1989). Thus, higher levels of Pablp in the cytoplasm may
lead to increased mRNA stability and a coincident upsurge in translation initiation
thereby alleviating the deleterious effects of the export block in the nab2A strain.
Arguing against this explanation, Pablp was found to be present in both the
nucleus and cytoplasm by immunofluorescence microscopy when PAB1 was
expressed from a high-copy plasmid (Hector et al., 2002).
Nuclear Targeting of Pablp Suppresses the nab2A Growth Defect
To prove Pablp is required in the nucleus to compensate for the loss of
Nab2p, DNA encoding an SV40 nuclear localization (NL) sequence (PKKKRKV)
and a mutant NL (mnl) sequence (PKTKRKV) was cloned 5’ of the PAB1 start
codon in a low-copy plasmid (pPAB1NL and pPAB1mni, respectively). In addition,
as a means of discriminating between endogenous Pablp and the Pab1NL or
Pablmni fusion proteins, the stop codon of PAB1 was replaced by sequence
encoding the V5 epitope (Invitrogen). These plasmids were transformed into the
na£>24pGAL::NAB2 shuttle strain (YRH201c). When grown on galactose at 24°
C, both the nat»2ztpPAB1NL and na£>2zlpPAB1mni strains grow (Figure 8A)
because Nab2p is still present (Figure 8B and data not shown). When cells were
streaked to glucose, only the na£>2ztpPAB1NL and NAB2 strains show growth
(Figure 8A). Importantly, the Pab1NL and Pab1mni proteins were expressed at

Figure 8. Targeting Pablp to the nucleus suppresses the nab2A growth defect. A) Suppression of nab2A by low-copy
pPAB1NL. Plate growth of NAB2, r?ab24pGAL::NAB2, nab2ApPAB']NL and nab24pPAB1mni strains on YPGal
(galactose) and YPD (glucose) at 24°C is shown. B) Immunoblot analysis of NAB2, nab2z\pPAB1NL and
na¿>2z\pPAB1mni strains grown in glucose and galactose. Proteins were detected using anti-V5 (aV5), anti-
Pablp 1G1 (aPablp) and anti-Nab2p 3F2 (aNab2p) antibodies.

A
B
Galactose
nab2A
pGAL::NAB2 pPAB1NL
nab2A
pGAL::NAB2
NAB2
nab2A
r'Ar'1NL
nab2A
mnl
nab2A
pPAB1 mn|
aPabl
aV5
aNab2
Glucose
glucose galactose
-'j
cn

76
levels similar to the endogenous Pablp as indicated by immunoblot analysis
using aPabl 1G1 (Figure 8B). Unlike endogenous Pablp, however, the Pab1NL
protein, as visualized by cell immunofluorescence using the aV5 antibody
(Invitrogen), accumulated in the nucleus (Figure 9A). Surprisingly, import of the
Pab1NL protein was incomplete (Figure 9A) suggesting either import by the SV40
NLS in yeast is relatively inefficient or PablNL is also rapidly exported.
Nevertheless, these results clearly demonstrate Pablp must cycle through the
nucleus to compensate for the loss of Nab2p.
Nab2p Is Required for Polv(A) Tail Length Control and mRNA Export
Flow does nuclear Pablp suppress the growth defect of a nab2A strain?
Since Pablp has been implicated in poly(A) tail trimming in vitro and in vivo
(Amrani et al., 1997; Brown et al., 1996; Minvielle-Sebastia et al., 1997), poly(A)
tail lengths were assayed in /iab24pPAB1NL (data not shown). Poly(A) tails were
actually 10-20 nucleotides longer than previously described for naib2zlpPAB1
(Hector et al., 2002) indicating, in the absence of Nab2p, targeting Pablp to the
nucleus is insufficient to limit poly(A) tail length in vivo. The localization of
poly(A)+ RNA in both the nab24pPAB1NL and nab2ApPAB'\ strains was also
determined by fluorescence in situ hybridization (FISH) using a d(T)50 probe. A
major portion of the poly(A)+ RNA signal was present in the cytoplasm of both
strains (Figure 9A, nab2ApPAB'\NL and nab2ApPAB'\ rows), although a
significant nuclear accumulation of poly(A)+ RNA was still observed (Figure 9A,
compare poly(A)+ RNA fluorescence in na£>24pPAB1Ni_ to that of NAB2).
Remarkably, nuclear accumulation of poly(A)+ RNA in nab2ApPAB'\NL cells

Figure 9. Pab1NL accumulates in the nucleus of nab2ApPAB'\NL. A) SV40 NLS-
tagged Pablp and poly(A)+ RNA concentrate in the nucleus of a nab2A
strain. The localizations of V5-tagged Pablp, poly(A)+ RNA and DNA
are compared in NAB2, nab2ApPAB'\Nl and na£>24pPAB1mni strains.
B) Suppression of low-copy pPAB1 NL is restricted to 24° C. Serial ten¬
fold dilutions of NAB2 and nab24pPAB1NL strains were spotted on
YPD plates. Plates were incubated for either 3 days (24° C and 37° C)
or 7 days (14° C).

78
A
aV5 poly(A)+ RNA DNA
NAB2
nab2A
pPAB1
nab2A
pPAB1NL
B
14ooC
24°°C
37ocC
NAB2
nai)2ApPAB1 nl
© ® * -

79
correlated with an inability to grow at high (37° C) and low (14° C) temperatures
(Figure 9B). Two competing hypotheses can be proposed to explain these
findings: (1) the accumulation of Pablp in the nucleus (Figure 9A) inhibits mRNA
export as well as growth at temperatures other than 24° C or (2) Nab2p functions
in some capacity to stimulate mRNA export and Pablp is unable to replace
Nab2p in this role. I favor the latter hypothesis because the RNA localization and
polyadenylation phenotypes of nab2ztpPAB1NL resemble those observed in
connection with mutations in known mRNA export factors. Poly(A) tail lengths
were assayed in the temperature-sensitive mRNA export mutant (mex67-5) and
the RanGAP temperature-sensitive mutant (rna1-1). A severe hyperadenylation
defect was observed in mex67-5 at the non-permissive temperature (37° C), but
not the permissive temperature (24° C), while rna1-1 only exhibited a slight
increase in poly(A) tail length at 37° C (Figure 10). Importantly, RNA1 is required
for protein import but not mRNA export (Koepp et al., 1996), whereas MEX67 is
required for efficient mRNA export but not protein transport (Segref et al., 1997).
Therefore, a tight correlation between nuclear mRNA export and poly(A) tail
length restriction was observed. These results parallel those described for
several other mRNA export mutant strains, including mtr2, dbp5, and rat7
(Flilleren and Parker, 2001; Jensen et al., 2001b). Jensen et al. (2001) reported
that hyperadenylated HSP104 transcripts accumulated in nuclear foci when rat7-
1 cells were shifted to non-permissive temperatures (Jensen et al., 2001b). The
nuclear exosome, in particular Rrp6p, is required for foci retention of both
hypoadenylated SSA4 RNA in pap1-1 and hyperadenylated HSP104 RNA in

Figure 10. Hyperadenylation and mRNA export defects correlate in mex67-5.
Poly(A) tail analysis of MEX67, mex67-5 and rna1-1 grown in YPD at
either 24° C or shifted to 37° for 30 minutes. Each lane represents
equal amounts of labeled RNA. The indicated size markers are
pBR322 Msp\ fragments.

81

82
rat7-1 (Hilleren et al., 2001). Intriguingly an accumulation of poly(A)+ RNA in
sub-nuclear foci was also observed for nab2AGAL::NAB2 when grown in glucose
for 2 hours and nab2-21 when shifted to 14° C for 30 minutes (Anderson, 1995;
Hector, 2000). Considering the nuclear exosome and Rrp6p is responsible for
processing 3’ ends of rRNA and snoRNA (Allmang et al., 1999a) and GFP-Rrp6p
localizes predominately to the nucleolus (Huh et al., 2003), it is possible that
hyperadenylated RNAs are trafficked to the nucleolus for retention and degraded
by the exosome when Nab2p is not functional. Furthermore, Pablp may allow
for growth when targeted to the nucleus in a nab2A strain by inhibiting nucleolar
retention of poly(A)+ RNA. Providing support for this model, poly(A)+ RNA
colocalized with an abundant nucleolar protein (Nopip) in both nab2AGAL::NAB2
and nab2-21 under non-permissive growth conditions (Figure 11) but poly(A)+
RNA was observed throughout the nucleus and cytoplasm in the nab24pPAB1NL
strain (Figure 9A). Thus in each nab2 mutant strain examined, the lethal growth
phenotype correlated with nucleolar localization of poly(A)+ RNA. Based on
these results I propose that Nab2p normally functions to inhibit nucleolar
retention, and possibly degradation, of polyadenylated RNA while at the same
time facilitates the export of poly(A)+ RNA to the cytoplasm. Targeting Pablp to
the nucleus inhibits nucleolar retention but does not restrict tail lengths or
promote mRNA export in the absence of functional NAB2. Further evidence is
needed to support the idea that Nab2p and Rrp6p have antagonistic roles in
nuclear mRNA export/retention and poly(A)+ RNA stabilization/degradation, but

Figure 11. Loss of nab2 function leads to nucleolar accumulation of poly(A)+
RNA in vivo. Co-localization of poly(A)+ RNA (green) and Nopip (red)
in nab2zipGAL:NAB2 cells shifted to glucose for 2 hours and nab2-21
cells shifted to 14° C for 30 minutes. FISH analysis of poly(A)+ RNA
distribution was performed using conventional immunoflourescence
microscopy. DNA was detected with DAPI.

poly(A)+RNA Nopip
merge/DAPI

85
this model also predicts that Nab2p recruits a nuclear export receptor to the
poly(A) tail which stimulates the nuclear export of mRNA.
Nab2p Associates with Factors Required for mRNA Export
Several recent studies have implicated Nab2p in mRNA export (Duncan et
al., 2000; Green et al., 2003; Green et al., 2002; Hector et al., 2002). Loss of
NAB2 in vivo or shifting mutant nab2 strains to non-permissive growth
temperatures results in rapid accumulation of poly(A)+ RNA in the nucleus
(Green et al., 2002; Hector et al., 2002). Intriguingly, Nab2p and poly(A)+ RNA
co-localize near nuclear pores in a mutant tom1 strain (Duncan et al., 2000) and
Nab2p interacts with a NPC-associated protein (Mlplp) in the yeast two-hybrid
system (Green et al., 2003). The authors of these studies propose that Nab2p
functionally interacts with export receptors and the NPC to mediate the export of
poly(A)+ RNA. In addition, examination of the Nab2p domain structure revealed
a nuclear export sequence (NES) near the N-terminus of Nab2p that was
required for efficient poly(A)+ RNA export (Marfatia et al., 2003).
Purification of Nab2pT-associated Proteins
The founding member of the RNA export factor (REF) family of proteins
(Yralp) binds mRNA in the nucleus and forms a bridge between the site of
transcription and nuclear pore through interactions with the nuclear export factor
Mex67p (Portman et al., 1997; Strasser and Hurt, 2000; Stutz et al., 2000;
Zenklusen et al., 2001). In metazoan organisms, REF/Aly, SR proteins and
hnRNPs all serve as adapters for mRNA export, channeling mRNA through the
nucleus to the NPC via interactions with TAP (Gallouzi and Steitz, 2001; Huang

86
et al., 2003; Longman et al., 2003; Luo et al., 2001; Moore and Rosbash, 2001;
Zhou et al., 2000). It seemed likely that Nab2p may also promote mRNA export
of poly(A)+ RNA in yeast by serving as an adapter protein, recruiting an export
receptor to the poly(A) tail. The tandem affinity purification (TAP) protocol
(Rigaut et al., 1999) was employed to purify proteins that specifically associate
with Nab2p.
By virtue of homologous recombination, the wild-type NAB2 stop codon
was replaced with sequences encoding a calmodulin binding protein (CBP) and
protein A (ProtA) to generate the NAB2T strain (YKN206) (Figure 12A). A
protein that migrated more slowly (Nab2pT) than the wild type protein (Nab2p)
was detectable by immunoblot analysis using aNab2p 3F2 (Figure 12B)
indicating recombination was successful. Importantly, levels of Nab2pT
corresponded to that of Nab2p (Figure 12B) and growth rates of the two strains
were comparable (data not shown). The dual CBP-ProtA tag allows for
purification of native Nab2pT, and any associated proteins, by two successive
steps (Figure 13). Briefly, whole cell extracts were prepared from YKN206 and
incubated at 4° C with IgG Sepharose (Pharmacia). The ProtA peptide has a
relatively high affinity for Immunoglobulin G (IgG) protein allowing for retention of
Nab2Tp-associated proteins (Nab2pTAP) on the Sepharose beads. After several
washes to remove nonspecific proteins, the addition of TEV released Nab2pTAP
from the IgG Sepharose by cleavage between the CBP and ProtA peptides (see
Figure 12A). The TEV eluate was subsequently incubated at 4° C with
calmodulin affinity resin (Stratagene). In this case, the CBP tag is indispensable

Figure 12. Genomic tagging of NAB2 with the CBP-ProtA tag. A) Replacement of the NAB2 stop codon with calmodulin
binding peptide (CBP) and protein A (ProtA) sequences by homologous recombination. Start and stop
codons for NAB2 and TRP1 genes and TEV protease site sequences are indicated. B) Detection of the
Nab2p and Nab2p-Tagged (Nab2pT) proteins using anti-Nab2p (3F2). Each lane represents equal amounts
of total protein isolated from NAB2 or A/AB2-tagged (NAB2T) strains.

A
ATG Stop
NAB2
214-
-Nab2pT
-Nab2p
44-
28-
18-
oo
00

89
because 2 mM CaCI2 induces binding of Nab2p to the calmodulin resin and 2
mM EGTA chelates the CaCI2 releasing Nab2pT and all associated proteins
(Nab2pTAP) under mild, non-denaturing, conditions.
Nab2p and Kap104 Form an Abundant Complex in Yeast Extracts
The Nab2pTAP was first used to generate antibodies against the most
abundant proteins. Standard procedures were performed to inject 5 pig of purified
proteins into BALB/c mice (Harlow and Lane, 1988). After an appropriate
immune response, the polyclonal antiserum was used to screen a Agt11 genomic
expression library as previously described (Snyder et al., 1987). Seventeen
phage clones positively reacted against the Nab2pTAP antiserum. Of these
clones, thirteen were also immunoreactive to aNab2 3F2 and aNab2 1F2. The
remaining clones were sequenced and discovered to encode for Nab2p (2
clones) and Kap104p (2 clones). Interestingly, both NAB2 clones initiated
translation near the QP region of Nab2p suggesting 3F2 and 1F2 recognize
sequences within the NES region of Nab2p (see Figure 14A). To explore this
possibility further, two N-terminal truncations (NT1 and NT2) of Nab2p (Figure
14A) were fused to the GAL4 activation domain (AD) peptide (Clontech) (Figure
14B) and tested for 3F2 and 1F2 immunoreactivity. Monoclonal antibody aNab2
1F2 recognizes only AD-NT2 while aNab2 3F2 recognizes both AD-NT1 and AD-
NT2 peptides (Figure 14C), restricting the 3F2 epitope to the first 42 amino acids
of Nab2p.
Kap104p, a member of the importin (3/karyopherin (3 family of transport
receptors, is responsible for importing both Nab2p and Nab4p into the nucleus

Figure 13. Purification scheme of Nab2pT and associated proteins. Whole cell protein extracts were prepared from the
NAB2T (YKN206) strain and incubated in the presence of IgG sepharose beads at 4° C. Release of Nab2pT
from the IgG column was accomplished by cleavage with the tobacco etch virus (TEV) protease. Calmodulin
affinity resin was used to bind TEV-released Nab2p-CBP in the presence of 2 mM CaCI2 at 4° C. Calmodulin-
bound proteins were eluted by the addition of 2 mM EGTA and fractionated by SDS-PAGE.

Whole Cell
Extract
Protein Identification
IgG Sepharose
Calmodulin Affinity Resin
+ 2 mM CaCl2
CO

92
(Aitchison et al., 1996). Kap104p interacts with RG-rich domains present in both
Nab2p and Nab4p and mediates import through additional interactions with
GLFG-repeat containing nucleoporins (Nup116, NuplOO, Nup145, and Nup57)
(Aitchison et al., 1996). Together, RanGTP and RNA abrogate the binding of
Kap104p to Nab2p and Nab4p, providing a mechanism for Kap104p-Nab2/4
dissociation once in the nucleus (Lee and Aitchison, 1999). In the cytoplasm,
hydrolysis of RanGTP is believed to release Nab2p and Nab4p from mRNA and
induce association with Kap104p for another round of import because Kap104p,
but not Kap95p, displaces both Nab2p and Nab4p from single stranded DNA
(ssDNA) cellulose (Lee and Aitchison, 1999). Nab4p is most similar to metazoan
hnRNP A1 and the Kap104p homologue (Trn 1) imports hnRNP A1 into the
nucleus of COS7 cells (Siomi et al., 1997). Although a metazoan protein with
significant similarity to Nab2p has not been described, the RGG-domain of
Nab2p interacts with Trn 1 in vitro and is sufficient for nuclear import of reporter
proteins in human cells (Siomi et al., 1998; Truant et al., 1998). Recently, the
Trn 1-hnRNP A1 heterodimer has also been implicated in the export of specific
mRNAs (Gallouzi and Steitz, 2001; Moore and Rosbash, 2001). However, a
direct role for the yeast Kap104p-Nab2p complex in mRNA export is unlikely
because kap104 mutants exhibit a delayed (> 3 hours) accumulation of poly(A)+
RNA in the nucleus (Aitchison et al., 1996). Nevertheless, discovery of Kap104p
immunoreactivty in the polyclonal antiserum suggests Kap104p is an abundant
component of the purified Nab2pTAP complex. To substantiate this claim, the

Figure 14. The epitopes of aNab2p antibodies map to the NES of Nab2p. A) The domain structure of Nab2p includes a
putative nuclear export sequence (NES), a glutamine-proline sequence (QP), an arginine/glycine-rich domain
(RGG) and seven cysteine-cysteine-cysteine-histidine (C3H) domains. The N-terminal portion of Nab2p was
truncated at NT1 and NT2 by PCR. B) NT1 and NT2 sequences were fused to the GAL4-activation domain
(GAL4-AD) open reading frame. C) Immunoblot analysis of AD-NT1 and AD-NT2 fusion proteins with cxNab2
3F2 and aNab2 1F2. Each lane represents equal amounts of total protein. Molecular weight markers are
indicated.

A
MSQEQYTENLKVIVAEKLAGIPNFNEDIKYVAEYIVLLIVNGGTVESWDELASLFDSVSRDTLANWQTAFFALEALQQG
B
GAL4-AD NT1
GAL4-AD NT2
C
I—3F2 —I
I—1F2 —I
CD

95
KAP104 phage clones were used to screen hybridoma cell lines for the isolation
of the aKap104p 1D12 monoclonal antibody.
Nab2p Co-ourifies with Nuclear mRNA-bindinq Proteins
When Nab2pTAP was partitioned on a 12.5% polyacrylamide-1 % SDS
gel, the aKap104 antibody detected a protein co-migrating with the 100 kDa
marker protein (Figure 15B). Coomassie blue staining of the eluate revealed a
complex array of proteins associated with Nab2pT, although Nab2p and the 100
kDa protein (Kap104p) are clearly the two most abundant proteins (Figure 15A).
While the amount of Kap104p purified with Nab2pT was not quantified, the
Kap104p-Nab2p heterodimer appears to be at least 10 percent of the total
purified protein content. Based on these results, the corresponding argument
can be made that a significant fraction of Nab2p is unable to bind RNA and forms
an RNA-independent complex with Kap104p in whole cell extracts. In support of
this idea, the majority of Nab2p was also immunodepleted by aKap104 from
cleavage and polyadenylation cell extracts (Ronald Flector, personal
communication). Given the affinity of Nab2p for poly(A) RNA (see Figure 5), we
speculated a second, RNA-dependent complex was also a constituent of
Nab2pTAP. As expected, poly(A)+ RNA was isolated from the calmodulin eluate
(data not shown) and several mRNA-binding proteins, including Pablp, Npl3p,
Mex67, and Yralp, were detected by immunoblot analysis (Figure 15).
Interestingly, neither Nab4p nor Nab3p were found to associate with Nab2pT in
protein extracts. Nab4p and Nab2p interact in the yeast two-hybrid system (Uetz
et al., 2000) and they appear to have antagonistic roles in polyadenylation

Figure 15. Nab2p associates with nuclear-cytoplasmic transport factors in protein extracts. A) Visualization of Nab2pT
purified proteins by coomassie blue stain. The calmodulin affinity column eluate was fractionated by SDS-
PAGE. Proteins were visualized by staining with Coomassie blue. Molecular weights are indicated. B)
Immunoblot analysis of Nab2pT purified proteins using monoclonal antibodies 3F2 (aNab2), 1D12 (aKap104),
1G1 (aPabl), 2F12 (cdMab3), 3H1 (aNab4) and polyclonal antibodies, aNpl3, aMex67, aYral.

A
250 -
100 -
75 -*
50 -
37 -
15 -
Coomassie
Immunoblots
CD
-nI

98
(Figure 6). Nab4p is recruited to RNAP II as a component of the cleavage and
polyadenylation factor, CF I (Barilla et al., 2001; Kessler et al., 1997) and binds to
AU-rich regions of the pre-mRNA specifying appropriate cleavage sites and
stimulating efficient polyadenylation (Minvielle-Sebastia et al., 1998). In contrast,
Nab2p interacts with the poly(A) tail, and possibly Nab4p, to terminate
polyadenylation. Nab3p associates with elongating RNAP II through interactions
with Nrdlp and the CTD (Conrad et al., 2000) and mediates the termination and
cleavage of non-polyadenylated small nuclear RNAs in conjunction with Ssu72p
(Dheur et al., 2003; Steinmetz and Brow, 2003).
To confirm the immunoblot analysis and identify other proteins that
interact with Nab2pT in vivo, Matrix Assisted Laser Desorption Ionization-Time Of
Flight Mass Spectrometry (MALDI-TOF MS) was performed in collaboration with
John Aitchison and David Dilworth at the Institute for Systems Biology (Seattle,
WA). After fractionation on a 4-20% SDS-PAA gel and visualization of the
proteins by silver stain, selected regions were excised and subjected to MALDI-
TOF MS (data not shown). A list of proteins relevant to Nab2p function can be
found in Table 1. In addition, components of both large and small ribosomal
subunits, translation initiation factors, heat shock proteins and GTP biosynthetic
enzymes (Imd2p-lmd4p) were also identified (data not shown) but we do not
believe these proteins functionally interact with Nab2p because they have been
reported to contaminate tandem affinity purifications (Gavin et al., 2002). Based
on the immunoblot and MS analysis, the Nab2pT-associated proteins can be
grouped into two functional classifications. The sole member of the first group is

99
TABLE 1.
Nab2pT-associated proteins
Name*
MW*
Localization*
Protein Description
Nuclear-Cytoplasmic Transport
Kapl04p
104
N; c
karyopherin P; imports receptor for Nab2p and Nab4p
Mex67p
67
N
nuclear mRNA export receptor
Npl3p
45
N
nuclear mRNA binding; mRNA biogenesis
Yralp
25
N
RNA annealing protein; mRNA export adapter
Gfdlp
21
N; C; NPC
mRNA export; stimulates Dbp5p activity in vivo;
RNA Processing
Poplp
100
N; C; NO
RNase P component; rRNA and tRNA processing
Nop56p
56
N; NO
nucleolar protein; rRNA biogenesis
Lsm6p
14
N
U6 snRNA-associated protein
*Saccharomyces Genome Data Base (SGD) standard gene name; www. yeastgenome. org
"Predicted Molecular Weight in kilodaltons (kDa); www. yeastgenome. org
* Yeast GFP Fusion Database at yeastgfp.ucsf.edu; N, nucleus; C, cytoplasm; NPC; nuclear pore
complex; NO, nucleolus; CW, cell wall; VM, vacuolar membrane; ND, not determined
# Yeast Proteome Database; www.incvte.com

100
Kap104p and most likely represents a functional Nab2p-Kap104p import
complex. The absence of detectable Nab4p suggests Kap104p imports Nab4p
and Nab2p separately in vivo. The second classification includes nuclear mRNA-
binding proteins (Mex67p, Yralp, and Npl3p). Npl3p has been proposed to bind
to pre-mRNA during transcription and package the mRNA into a complex
competent for export (Krebber et al., 1999; Lee et al., 1996; Lei et alM 2001;
Shen et al., 2000). Yralp also associates with pre-mRNA during transcription as
a member of the TREX complex (Strasser et al., 2002; Zenklusen et al., 2002).
Importantly, Sub2p is also a member of the TREX complex and Yra1p-Sub2p
interactions must be disrupted before the Yra1p-mRNA is released from the site
of transcription and exported to the cytoplasm by Mex67p (Strasser and Hurt,
2001). Because proteins known to function predominately at the site of
transcription (Sub2p, Nab4p, and Nab3p) did not co-purify with Nab2pT it is likely
that we have purified a Nab2p-mRNP that has been released from the TREX
complex and is in the process of being exported. These results also predict that
interactions between Nab2p and Mex67p, Yralp or Npl3p may be important for
mediating the export of poly(A)+ RNA to the cytoplasm after release from
chromatin.
mRNA Export Is Inhibited by a Mutation in the NES of Nab2p
Several years ago, pNAB2-20 was isolated from a plasmid library that
when shuttled into YJA501 (nab2ApGAL::NAB2) conferred cold-sensitive and
temperature-sensitive growth phenotypes (Anderson, 1995). The NAB2 genetic
defect was subsequently determined to result in a leucine to proline (L -> P)

101
mutation near the N-terminus (Anderson, 1995) (also see Figure 16A). The
pNAB2-20 plasmid has recently been transformed into a nab2ApGAL::NAB2
strain with a different genetic background (YRH201c) and exhibits only a
temperature-sensitive phenotype (YKN220) (Figure 16B). FISH analysis using a
d(T)5o probe revealed an accumulation of poly(A)+ RNA in the nucleus of nab2-20
cells at both 24° C and 37° C (Figure 16C) very similar to the mRNA export block
observed for nab2ApPAB1NL (Figure 9A). Why is poly(A)+ RNA blocked for
export in nab2-202 Considering the N-terminus of Nab2p contains a putative
NES (Marfatia et al., 2003), it seemed likely the L -> P mutation of nab2-20 alters
the affinity of Nab2p for an export receptor resulting in an accumulation of
poly(A)+ RNA in the nucleus. In order to purify and identify proteins that
specifically interact with the N-terminus of Nab2p, the tandem affinity purification
procedure was once again utilized.
Mex67p Interacts with the N-terminus of Nab2p
The first 82 amino acids of Nab2p (NT2; Figure 14A) was fused to CBP
and ProtA peptides (NT2pT) and expressed in a wild type strain. In addition, a
Nab2-20p NT2 TAP-tagged peptide (nt220pT) was also generated and served as
a control for the NT2pT purification. Neither NT2pT nor nt220pT adversely
affected growth when expressed from a low copy plasmid, although both
peptides were less abundant than endogenous Nab2p (data not shown). Silver
staining of the NT2pT-associated proteins (NT2pTAP) revealed very few proteins
above background (Figure 17A). However, we observed a pair of stained bands
with intensities similar to the NT2pT peptide(s) and apparent molecular weights

Figure 16. Constitutive nuclear accumulation of poly(A)+ RNA in nab2-20. A) A
leucine to proline mutation (L->P) leads to temperature-sensitivity in
nab2-20. B) Spot plate growth phenotypes of nab2-20. Serial ten¬
fold dilutions were grown on YPD plates at 14° C for 7 days or 24°
and 37° C for 3 days. C) FISH analysis of poly(A)+ RNA distribution
in NAB2 and nab2-20 at 24° and 37° C. Cell morphology was
visualized by differential interference contrast (DIC) microscopy.
DNA was detected with DAPI.

103
A
Nab2-20p
Nab2p
o° ^
O^cT5
c^or
(S’ &
£
>w-*w-w-
B
NAB2
nab2-20
14ooC 24°oC 37°°C
C
NAB2
24°oC
NAB2
37ooC
nab2-20
24ooC
nab2-20
37ooC
poly(A)+ RNA DNA DIC

104
of ~65 kDa and ~40 kDa (Figure 17A, closed stars). Kap104p binds to the RGG-
domain of Nab2p, and as expected did not co-purify with the N-terminus (Figure
17A). Intriguingly, Mex67p co-purified with NT2pT as demonstrated by
immunoblot analysis using an aMex67 polyclonal antibody (gift from Ed Hurt,
University of Heidelberg, Heidelberg, Germany). RNA tethering between NT2pT
and Mex67p was ruled out by the addition of RNaseA (200 pg/ml) to extracts
during purification (Figure 17A). In addition we did not detect any interactions
between NT2pT and either Npl3p, Pablp or Yralp (Figure 17A). The silver stain
pattern of the nt220pT-associated proteins (nt220pTAP) was quite different from
that of NT2pTAP (compare Figure 17A, silver stain to Figure 17B, silver stain). A
single intense band migrated between 50 and 37 kDa and a cluster of bands co¬
migrated with the 25 kDa marker protein (Figure 17B, open stars). In contrast,
the intensity of the 67 kDa band was profoundly reduced (Figure 17B).
Furthermore, immunoblot analysis indicated both Mex67p and Pablp interact
with nt220pT in vivo (Figure 17B). The addition of RNase A during purification of
nt220pTAP ruled out the possibility that RNA mediates either Mex67p-nt220pT or
Pab1p-nt220pT interactions (Figure 17B). To eliminate the possibility that other
yeast proteins were responsible for Mex67p-Nab2p interactions, a GST pull¬
down assay was performed.
nab2-20 Stabilizes Mex67p-Nab2p Interactions In Vitro
Both Mex67p and TAP contain a conserved protein-protein interaction
motif characterized by Leucine Rich Repeats (LRR) (Liker et al., 2000; Segref et
al., 1997). LRRs typically form a horseshoe-like structure with (3-sheets forming

Figure 17. Interactions between the N-terminus of Nab2p and Mex67p. A) Silver stain and immunoblot analysis of Nab2-
NpT purified proteins. Proteins enriched by tandem purification of the Nab2-NpT are indicated (closed star).
RNase treatment (+) was performed by the addition of RNase A (200 pg/ml) to protein extracts prior to
purification. B) Silver stain and immunoblot analysis of Nab2-20-NpT purified proteins. Proteins enriched by
purification of Nab2-20-NpT are also indicated (open stars). Molecular weight standards are indicated.

A
Antibody
RNase
250-
100-
37- ’♦
25-
20-
15-
Silver L
Immunoblots
NAB2-N CBP ProtA
250-
100-
75-
50-
37-
25--?«
20-
15
Silver
Immunoblots
nab2-20-N cbp ProtA
CL
o
CD

107
an inner concave surface and a-helices on the outside (reviewed in Kobe and
Kajava, 2001). Typically, ligand-binding sites for LRR proteins map to exposed
residues on the inner surface of the LRR. In the case of Mex67p and TAP, the
LRR is responsible for binding to mRNA export adapter proteins (Huang et al.,
2003; Strasser and Hurt, 2000). Interestingly, the N-terminus of Yralp contains
a short non-canonical leucine-rich region that is required for interactions with
both Mex67p and Sub2p (Strasser and Hurt, 2001). Closer examination of the
Nab2p N-terminus revealed a short leucine-rich region surrounding the L->P
mutation that was remarkably similar to the Yra1 N-terminus (Figure 18A). The
LRR of Mex67p (see Figure 18A) was fused in-frame to a GST protein open
reading frame and the GST-LRR was expressed in E. coli cells. Bacterial lysates
were prepared and GST-LRR was immobilized on glutathione-Sepharose
(Pharmacia). Similar portions of GST-LRR bound Sepharose was incubated with
lysates prepared from bacterial strains expressing the N-terminus of Nab2p
(Nab2pN) and Nab2-20p (Nab2-20pN). After binding and washing, the
precipitated proteins were denatured and immunoblots were performed using
aNab2 1F2. Although aNab2 3F2 recognizes the first 42 amino acids of Nab2p
(see Figure 13), it was unable to detect the N-terminus of Nab2-20p (data not
shown) so it was not used for these studies. Astonishingly, more Nab2-20pN
precipitated with GST-LRR than Nab2pN (Figure 14B, compare Nab2pN to
Nab2-20pN) suggesting the L->P mutation stabilizes the Nab2p-Mex67p
interaction. Since the expression levels of Nab2pN and Nab2-20pN in bacteria
are not significantly different (Figure18B, lysate lane) and identical lysate

Figure 18. The nab2-20 mutation stabilizes the interaction between Nab2p and
the LRR of Mex67p in vitro. A) A leucine rich in the N-terminus of
Nab2p (Nab2) is similar to a comparable region of Yra1 p (Yra1). The
L->P mutation is indicated (Nab2-20). The leucine rich repeat (LRR)
of Mex67p was fused to glutathione S-transferase for purification
purposes. Amino acid numbers for Nab2p and Mex67p are
indicated. B) Lysate of bacterial strains expressing Nab2pN and
Nab2-20pN peptides were incubated at 4° C with glutathione beads
pre-bound with GST-LRR, GST-Pab1, GST-Sub2, GST-Yra1, GST-
Npl3, or GST. Nab2pN Nab2-20pN were detected using aNab2 1F2.

109
A
Mex67p
1 264 600
'—
LRR
C l
GST
â– 
LRR
Yra1
Nab2
Nab2-20

110
volumes were added to GST-LRR beads, the input of Nab2pN and Nab2-20pN
proteins was deemed similar for both experiments. In addition to GST-LRR,
Nab2pN and Nab2-20pN peptides also interacted with GST-Pab1p and GST-
Sub2p proteins. Preferential interaction with Nab2-20pN was not as profound for
GST-Pab1p or GST-Sub2p (Figure 18B, compare Nab2pN to Nab2-20 in each
case). Importantly, neither Nab2pN nor Nab2-20pN interacted with GST-Yra1p,
GST-Npl3p, or GST (Figure 18B). From these results we conclude that Nab2p
and Mex67p physically interact in vitro and the L->P mutation appears to stabilize
this interaction.
Nab2p Is Phosphorvlated in Strains Defective for mRNA Export
Why does the N-terminus of Nab2p interact with Mex67p in vivo, but
interacts poorly with the LRR of Mex67p in vitro? The LRR may not fold correctly
in bacterial cells or Nab2p may require other regions of Mex67p, in addition to
the LRR, for binding. Alternatively, a post-translation modification of Nab2p may
be required for the Nab2p-Mex67p interaction in vivo. The export of U snRNA
requires cap-binding by the CBC, nuclear pore association by CRM1/RanGTP,
and a phosphorylated adapter (PHAX) to bridge the interactions between CRM1
and the CBC. A ternary complex of RNA, CBC, and PHAX readily forms in vitro,
but CRM1 only associates with the U snRNA export complex if PHAX has been
phosphorylated (Ohno et al., 2000). Although the phosphorylation sites have not
been mapped for PHAX, a leucine-rich NES, reminiscent of the HIV REV NES,
was identified and demonstrated to be essential for interactions between CRM1
and PHAX. How does phosphorylation promote a PHAX-CRM1 interaction?

111
Phosphorylation may induce a conformational change of the normally masked
NES or CRM1 may preferentially interact with the phosphorylated NES (p-NES).
Intriguingly, aNab2 1F2 detected two distinct NT2pT-purified proteins (Figure
17A). It is possible that NT2p-CBP has been partially cleaved although this
hypothesis is not favored because nt220p-CBP migrates as a homogeneous
band (Figure 17B). Alternatively, a portion of the NT2pT peptide may have a
post-translational modification. Studies are currently under way to determine if
the slower migrating NT2pT isoform is phosphorylated. Nevertheless, to
determine if phosphorylation of Nab2p correlates with Mex67p function, we
examined the phosphorylation status of Nab2p in mex67-5 cells (Figure 19A).
Total protein extracts were prepared from mex67-5 and the isogenic background
strain (MEX67) and then fractionated on an 8% polyacrylamide-1 % SDS gel. A
second, more slowly, migrating isoform of Nab2p correlated with the growth
defect of mex67-5 at 37° (Figure 19A). A slight increase in the upper isoform
was also noted for MEX67 at 37° C, but not to the same degree (Figure 19A).
Lamda phosphatase, when added to the mex67-5 protein extract, completely
eliminated the upper isoform (Figure 15B) indicating that Nab2p is
phosphorylated in mex67-5 extracts. Importantly, phosphorylation of Nab2p
does not affect RNA-binding activity because both isoforms crosslink to RNA
when subjected to UV light (Figure 19C). The phosphorylation status of Nab2p
was also analyzed in a second mRNA export mutant (dbp5-1) and the RanGAP
mutant (rna1-1) strains. In short, p-Nab2p was only observed in dbp5-1 (Figure
19A) cells once again correlating with an mRNA export block. Poly(A)+ RNA has

Figure 19. Phosphorylated Nab2p accumulates in mRNA export mutants. A) Increased concentrations of a higher
molecular weight aNab2 3F2 immunoreactive isoform in mex67-5 (37° C) and dpb5-1 (24° and 37° C) protein
extracts. No differences were oserved for Pablp (aPabl) or Npl3p (aNpl3) in any of the strains, at any
temperature tested. B) The upper isoform of Nab2p is phosphorylated. Treatment of extracts with A.
phosphatase (right lane) eliminated the higher molecular weight isoform. C) Both Nab2p isoforms are
intimately associated with RNA. Cellular proteins were covalently crosslinked by UV irradiation to RNA,
denatured, and purified by oligo(dT)-cellulose chromatography. Nab2p was visualized by immunoblot analysis
using mAb 3F2. Total protein extract without UV irradiation served as the control (left lane).

aPabl
cxNpl3
aNab2
B
C
*v
❖
ir
%
CO

114
been demonstrated to accumulate in the nucleus of dbp5-1 at both permissive
(24° C) and non-permissive temperatures (37°) (Tseng et al., 1998), whereas
rna1-1 directly affects protein import but not mRNA export (Corbett et al., 1995).
Based on these results, it is possible that phosphorylation of Nab2p stimulates
Mex67p binding to the NES. This model adequately explains the weak binding of
the LRR by Nab2pN in vitro. Furthermore, the proline mutation of Nab2-20p may
induce a conformation change of the N-terminus, constitutively exposing the NES
to Mex67p and prohibiting normal regulation of the Nab2p-Mex67p interaction by
phosphorylation. Future studies will be directed toward the isolation of high-copy
suppressors of nab2-20 and the mapping of Nab2p phosphorylation site(s) in an
attempt to demonstrate Nab2p and Mex67p functionally interact to mediate the
export of mRNA.

DISCUSSION
During synthesis, pre-mRNA is processed at the 3’ end to generate a
polyadenylated mRNA. Efficient cleavage and polyadenylation requires the
concerted action of several multi-subunit factors (Wahle and Ruegsegger, 1999).
Termination of polyadenylation generally yields an mRNA with a poly(A) tail
length of -70-90 nucleotides in yeast. After polyadenylation, the mRNA is
released from chromatin-associated RNAP II and exported to the cytoplasm.
Recent reports indicate that cleavage and polyadenylation events are intricately
connected to export of the mRNA. Inefficient release from RNAP II, as observed
in mRNA export mutants, leads to hyperadenylated mRNA (tail lengths > 150 nts)
(Hilleren and Parker, 2001; Jensen et al., 2001b). Alternatively, inefficient
polyadenylation, due to pap1 mutations, results in nuclear accumulation of
hypoadenylated mRNA (Hilleren et al., 2001). Is there a protein that functions in
polyadenylation, chromatin release and mRNA export which may be responsible
for the coordination of these events? Based on the observations presented in
this report, we believe this protein to be the nuclear poly(A)-binding protein
Nab2p.
Nab2p Is Required for Polv(A) Tail Length Restriction
Previous studies have suggested that Pablp is required for
polyadenylation in vivo since it shortens poly(A) tails to 60 nucleotides in vitro
115

116
and associates with CF IA in extracts (Amrani et al., 1997; Minvielle-Sebastia et
al., 1997). Deadenylation of hyperadenylated RNA by Pablp requires PAN
activity associated with the Pan2p-Pan3p complex (Boeck et al., 1996; Brown et
al., 1996). I propose that Nab2p, but not Pablp or Pan2p-Pan3p, limits poly(A)
tail lengths in vivo for the following reasons: (1) Nab2p binds strongly to poly(A)
RNA; (2) Nab2p localizes to the nucleus where it can bind to the poly(A) tail
during synthesis (Anderson et al., 1993b); (3) targeting Pablp to the nucleus did
not restrict poly(A) tail lengths in the absence of Nab2p; (4) when added to
pab1A extracts, Nab2p stimulates polyadenyation while restricting poly(A) tail
lengths; (5) at the earliest time points after addition of Nab2p to pab1A extracts,
poly(A) tails were found to be 90 nucleotides and deadenylation activity was not
observed. These observations suggest a model in which Nab2p binds to the
nascent poly(A) tail, stimulates efficient polyadenylation, and terminates
polyadenylation after the addition of ~90 adenosines (Figure 20). In the
cytoplasm, Pablp displaces Nab2p from the poly(A) tail and recruits Pan2p-
Pan3p which shortens poly(A) tails another 20-30 nucleotides.
Why is Nab2p activity inhibited in pab1A extracts? Nuclear import of
Nab2p requires Kap104p (Aitchison et al., 1996). The Kap104p-Nab2p
heterodimer has little affinity for ssDNA, and presumably RNA, while high levels
of RanGTP and RNA inhibit Kap104p-Nab2p interactions (Lee and Aitchison,
1999). To ensure that formation of the Nab2p-Kap104p complex occurs in the
cytoplasm and dissociation occurs in the nucleus, hydrolysis of RanGTP is
stimulated by a cytoplasmic GTPase activating protein (Rnalp) and GDP is

Figure 20. Model for poly(A) tail length restriction. Nab2p associates with the poly(A) tail during synthesis and terminates
polyadenylation after the addition of ~90 adenosines. In contrast, Pablp remains in the cytoplasm and
deadenylates poly(A) tails with Pan2p-Pan3p after export. These results predict that exchange between
Nab2p and Pablp is critical for cell viability. See text for details.

00

119
exchanged for GTP by a nuclear exchange factor (Prp20p) (Aebi et al., 1990;
Akhtar et al., 2001; Corbett et al., 1995; Dahlberg and Lund, 1998; Feng et al.,
1999). Extract preparation leads to mixing of nuclear and cytoplasmic
compartments, allowing the more abundant Rnalp protein (52,000 molecules/cell
vs. 12,000 molecules/cell for Prp20p [Huh et al., 2003]) to hydrolyze GTP more
efficiently than GDP can be exchanged for GTP. The result is an artificial
increase in Kap104p-Nab2p complex formation in protein extracts. In support of
this hypothesis, aKap104p 1D12 immunodepletes most of Nab2p from cleavage
and polyadenylation extracts (Ronald Hector, personal communication). Thus,
Kap104p inactivates Nab2p by inhibiting RNA-binding. Interestingly, low
RanGTP concentrations also correlate with hyperadenylation defects in vivo
because poly(A) tails are greater than 150 nucleotides in prp20 mutant strains
(Forrester et al., 1992) but are only 90 nucleotides in rna1-1 (see Figure 5).
An important question that emerges from the in vitro studies is the
mechanism of Nab2p-dependent poly(A) tail length control. The affinity of Nab2p
for r(A)25 suggests that Nab2p binds directly to the poly(A) tail. Furthermore,
inhibiting RNA-binding activity of Nab2p either by Kap104p in extracts or by
deleting a portion of the Nab2p RNA-binding motifs in vivo (Anderson et al.,
1993b; Hector et al., 2002) leads to hyperadenylation defects. Interestingly, tail
length control by Nab2p in vitro resembles that of PABPN1. Nab2p and PABPN1
both stimulate the conversion of oligo(A) precursors to poly(A) RNA at low
concentrations but inhibit polyadenylation at high concentrations. An appealing
model is that Nab2p stimulates polyadenylation by converting Paplp into a

120
processive enzyme through interactions with CF IB (Nab4p), the nascent poly(A)
tail, and Paplp. Nab2p and Nab4p have been demonstrated to interact in the
two-hybrid system (Uetz et al., 2000) and Nab4p is required for polyadenylation
in vitro (Kessler et al., 1997; Minvielle-Sebastia et al., 1998). However,
interactions between Nab2p and Paplp have not been described to date.
Because PABPN1 also stimulates polyadenylation in yeast extracts, it is unlikely
that direct interactions between Nab2p and Paplp are required for efficient
polyadenylation. Studies are currently underway to determine if human PABPN1
complements a nab2A strain and stimulates polyadenylation in yeast cells as
well.
After the addition of ~90 adenosines, Nab2p switches roles and terminates
polyadenylation. Remarkably, the addition of bovine PABPN1 to yeast extracts
also terminates polyadenylation at ~90 nucleotides arguing that: (1) maximum tail
length in a given organism is determined by the activity of their respective poly(A)
polymerase; (2) PABPN1 and Nab2p employ similar mechanisms for limiting
poly(A) tail lengths in vitro. Wahle and colleagues have proposed a PABPN1
counting mechanism for tail length control based on evidence correlating the
maximum size of an oligomeric PABPN1 with the maximum tail length of 250-300
nucleotides (Keller et al., 2000; Wahle and Ruegsegger, 1999). It is plausible
that Nab2p binds 20-30 adenosines and forms an oligomer on the poly(A) tail in a
manner similar to PABPN1. Alternatively, a single Nab2p molecule may bind 70-
90 adenosines, with each C3H motif associating with 10-15 nucleotides. Once
bound to the poly(A) tail, termination of polyadenylation in vivo may be triggered

121
by a post-translational modification of Nab2p which disrupts protein-protein
interactions or by the recruitment of an ATP-dependent RNA:protein unwindase.
Intriguingly, phosphorylation of Nab2p correlates with an accumulation of
hyperadenylated RNA in m RN A export mutants. A
phosphorylation/dephosphorylation cycle may be required for poly(A) tail length
restriction. In addition, we have demonstrated that Nab2p interacts with a
DExD/H-box helicase (Sub2p) in vitro. Sub2p has been postulated to promote
mRNA export at an early step after polyadenylation (Jensen et al., 2001a).
Nab2p may be phosphorylated by a CTD-associated kinase after binding to the
nascent poly(A) tail which recruits Sub2p and stimulates the release of Nab2p
from the polyadenylation machinery. Clearly, more experiments are needed to
tease apart the mechanisms involved in poly(A) tail length restriction by Nab2p in
vivo.
Potential Role for Nab2p in Preventing Nucleolar Retention of mRNA
Considering Nab2p is essential for cell viability and limits poly(A) tail
lengths in vitro, we were surprised to find that hyperadenylated mRNAs did not
adversely affect growth of nab2-21 (Hector, 2000; Hector et al., 2002). Even
more remarkable was the discovery that increased expression of PAB1
suppressed the nab2A growth defect but not the hyperadenylation defect (Hector,
2000; Hector et al., 2002). What is the essential, non-redundant, function of
Nab2p in yeast? Observations reported here suggest Nab2p may be required for
preventing the accumulation of poly(A)+ RNA in the nucleolus. In two nab2
mutant strains, colocalization of poly(A)+ RNA and Nopip correlated with loss of

122
viability. In contrast, restoration of viability correlated with nuclear and
cytoplasmic distribution of poly(A)+ RNA in na£>24pPAB1NL- Interestingly, Rrp6p
also colocalizes with Nopip in the nucleolus (Huh et al., 2003) and is required for
maturation of the 5.8S rRNA (Allmang et al., 1999a; Allmang et al., 1999b; Briggs
et al., 1998; Mitchell et al., 1997; Phillips and Butler, 2003), nuclear retention of
aberrant mRNA (Hilleren et al., 2001; Libri et al., 2002; Zenklusen et al., 2002),
and degradation of normal mRNA in the nucleus (Das et al., 2003). We propose
that Nab2p binds to the poly(A) tail during synthesis and normally prevents Rrp6p
and the exosome from retaining and degrading polyadenylated RNA in the
nucleus (Figure 21). In the absence of Nab2p, hyperadenylated RNA is retained
in the nucleolus. Importantly, hyperadenylation alone does not trigger nucleolar
retention because hyperadenylated RNA does not accumulate in nucleolar foci in
nab2-21 at permissive growth temperatures (Hector et al., 2002).
Why are hyperadenylated RNAs retained in the nucleolus in nab2
mutants? Burkhard and Butler (2000) identified rrp6-1 as an extragenic
suppressor of the pap1-1 strain. In the same study, they demonstrated physical
interactions between Npl3p and Rrp6p. Npl3p genetically interacts with the
nuclear yCBC and is recruited to the pre-mRNA during transcription elongation
(Lei et al., 2001; Shen et al., 2000). Npl3p and Rrp6p may associate with the
yCBC and retain the pre-mRNA at the site of transcription until polyadenylation
has been completed. If Nab2p is absent (nab2A) or unable to bind the poly(A)
tail (nab2-21), hyperadenylated mRNA may be shuttled to the nucleolus for
degradation by the exosome. If Nab2p binds to the poly(A) tail (NAB2) or Pablp

Figure 21. An integrated model for Nab2p function in polyadenylation, exosome release, and mRNA export. Binding of
Nab2p to the poly(A) tail serves to inhibit recruitment of the mRNA to the nucleolus, and possibly degradation,
by Rrp6p and the exosome. Nab2p, through interactions with Mex67p and the poly(A) tail, stimulates mRNA
export. See text for details.

124

125
is targeted to the nucleus (nab2ápPAB1NL), interactions between Rrp6p and the
Npl3p-CBC-mRNP may be disrupted. The C-terminus of Npl3p has been
demonstrated to interact with Nab2p (amino acids 8-524) in the two-hybrid
system (Inoue et al., 2000) and may functionally interact to inhibit Rrp6p binding.
Alternatively, interactions between Sub2p and Nab2p during polyadenylation may
stimulate rearrangement of the mRNP in a manner that inhibits interactions with
Rrp6p and stimulates release from RNAP II. Libri et al. (2002) have observed
Rrp6p-dependent degradation of HSP104 mRNA in sub2 mutant strains (Libri et
al., 2002) and we have demonstrated in this report that Nab2p and Sub2p
interact in vitro. Intriguingly, Sub2p interacts with Yralp and the THO proteins
(Tho2p, Hprlp, Mftlp, and Thp2p) during transcription and together the TREX
complex stimulates transcription elongation and splicing (Jimeno et al., 2002;
Kistler and Guthrie, 2001; Libri et al., 2001Strasser, 2002 #176). After, splicing
and polyadenylation of the pre-mRNA, Yralp must be released from the TREX
complex so that Mex67p can be recruited to the mature mRNP (Jensen et al.,
2001a; Strasser and Hurt, 2001; Zenklusen et al., 2002). NAB2 has recently
been isolated as a high-copy suppressor of thp1A and the nab2-1 strain
conferred transcription elongation defects similar to that of THO mutants
(Gallardo et al., 2003). Likewise, high-copy SUB2 suppresses elongation defects
associated with deletion of hpr1 (Jimeno et al., 2002) suggesting that Nab2p and
Sub2p both associate with elongating RNAP II and play similar roles in
stimulating transcription elongation. Since Mex67p, but not Sub2p, co-purifies
with Nab2pT we propose that Nab2p remains associated with the mRNA export

126
complex after it has been released from the TREX complex (see Figure 21).
Furthermore, direct interactions between Sub2p and Nab2p after polyadenylation
may be required for exchanging TREX components for mRNA export factors.
Intriguingly, partial deletion of the C3H RNA-binding motifs confers cold-sensitivity
in the nab2-21 strain (Hector et al., 2002) but a complete deletion of the C3H
motif domain results in cell death (Anderson, 1995; Marfatia et al., 2003)
suggesting that RNA-binding activity of Nab2-21p in vivo is not completely
compromised. Moreover, poly(A) tails are hyperadenylated in nab2-21 at the
permissive temperature (24°) (Hector et al., 2002) but poly(A)+ RNA does not
accumulate in the nucleolus until cells are shifted to 14°. In a recent study of
SUB2 function, 6 mutants were isolated and all of them exhibited cold-sensitive
phenotypes leading the authors to propose that Sub2p is critical for the
rearrangement of macromolecular complexes in vivo (Kistler and Guthrie, 2001).
Based on these findings, we propose that Sub2p function is critical for
rearrangement of the Nab2p-mRNP after polyadenylation but before mRNA
export. Experiments testing for genetic interactions between SUB2 and nab2-21
are needed to support this hypothesis.
Nab2o Interacts with Import and Export Receptors
The tandem affinity purification (TAP) scheme, as first described by
Bertand Seraphin and colleagues (Rigaut et al., 1999), has allowed for the
purification and comprehensive characterization of many functional yeast
complexes including the 60S pre-ribosome export complex (Bassler et al., 2001;
Nissan et al., 2002) and the cleavage and polyadenylyation complex (Dheur et

127
al., 2003; Gavin et al., 2002). By using the TAP scheme, we have purified a
Nab2p-Kap104p complex from yeast extracts. Several pieces of evidence
suggest that this represents a functional, RNA-independent heterodimer.
Kap104p interacts with the RGG-domain of Nab2p in vitro and Kap104p inhibits
binding of Nab2p to RNA (Lee and Aitchison, 1999). Nab2p activity is inhibited in
polyadenylation extracts and aKap104p 1D12 immunodepletes the majority of
Nab2p from these extracts. Visual inspection of the Nab2pT purified proteins by
Coomassie blue stain reveals Kap104p to be the most abundant protein besides
Nab2p. However, we do not believe that most of Nab2p is normally associated
with Kap104p in vivo because Nab2p is a nuclear RNA-binding protein
(Anderson et al., 1993b) and Kap104p is predominately cytoplasmic (Maurice
Swanson, personal communication). Protein extract preparation combines
nuclear and cytoplasmic compartments, which most likely leads to an artificial
increase in RanGDP concentration and promotes Kap104p-Nab2p interactions.
Purification of yeast nuclei could solve this problem, although this is an extremely
difficult task in yeast (John Aris, personal communication). The purification of
Nab2pTAP from a RanGAP mutant strain could also provide a solution. However,
rna1-1 is a temperature-sensitive strain and would require a shift to 37° C,
possibly altering the composition of Nab2p complexes as well.
Nab2pT also co-purified a nuclear mRNA export factor (Mex67p).
Although the abundance of Mex67p was much less than Kap104p or Nab2p, we
have provided evidence that interactions between Mex67p and Nab2p may be
important for mRNA export in vivo. We identified a leucine-rich sequence within

128
the NES domain of Nab2p that when mutated (nab2-20) results in nuclear
accumulation of poly(A)+ RNA. The N-terminus of Nab2p was subsequently
shown to physically interact with Mex67p and the nab2-20 mutation appears to
stabilize these interactions in vitro. Interestingly, the N-terminus of Yralp also
contains a leucine-rich sequence that interacts with Mex67p in vitro and is
required for mRNA export in vivo (Strasser and Hurt, 2001). Unfortunately,
several attempts to demonstrate that MEX67 and NAB2 genetically interact were
unsuccessful. Increasing the expression of MEX67 did not result in suppression
of the nab2-20 phenotypes (data not shown). Based on the in vitro pull-down
experiments, this result was not unexpected because the L->P mutation
increases binding of the Nab2p N-terminus to the LRR of Mex67p. We also
attempted to test for interactions between nab2-20 and mex67-5. However, each
time nab2-20mex67-5 diploids were sporulated a 2:2 (growth to no-growth) spore
ratio was observed (data not shown). Experiments involving MEX67nab2-20,
DBP5nab2-20, dbp5-1 nab2-20, and dbp5-2nab2-20 diploids yielded similar
results indicating that nab2-20 spores were not able to germinate after meiosis.
In addition to an mRNA export defect, nab2-20 cells are also greatly enlarged in
comparison to wild-type cells (see Figure 16C). Checkpoint genes acting
throughout the mitotic cell cycle govern cell size in Saccharomyces cerevisiae by
regulating growth and cell division (Boye and Nordstrom, 2003; Rupe, 2002).
Mutations in the SWE1 gene decrease cell size by inducing premature entry into
mitosis (Harvey and Kellogg, 2003). SWE1 also serves as a checkpoint during
meiosis by inhibiting nuclear division in cells that fail to complete meiotic

129
recombination and chromosome segregation (Leu and Roeder, 1999; Roeder
and Bailis, 2000; Wittenberg and La Valle, 2003). Given the connection between
meiosis and mitosis, it is not unexpected that nab2-20 cells, in addition to been
grossly enlarged, fail to grow after sporulation. Interestingly, connections
between Nab2p and the mitotic cell cycle have been made in the literature.
Mutations in tom1 (trigger of mitosis 1) lead to mislocalization of Nab2p and
poly(A)+ RNA in the nucleus (Duncan et al., 2000). Kap104p is somehow
responsible for the M-G1 transition because kap104-E604K bypasses the
requirement of the cyclin-dependent kinase gene (CDC15) for the exit from
mitosis (Asakawa and Toh-e, 2002).
We also decided to explore the possibility that Nab2-20p and Mex67p
form a hyperstable export complex in vivo. Dbp5p has been postulated to
remove nuclear proteins from recently exported mRNPs in the cytoplasm (Linder
and Stutz, 2001). GFD1 was initially identified as a high-copy suppressor of a
mutant dbp5 allele (rat8-2) and is believed to stimulate the remodeling activities
of Dbp5p in vivo (Hodge et al., 1999). Because Gfdlp interacts in the two-hybrid
system with Nab2p and co-purifies with Nab2pT (see Table 1), we reasoned that
increased expression of GFD1 and/or DBP5 in nab2-20 would stimulate
dissociation of the Nab2-20p-Mex67p complex and suppress the growth defects.
High-copy plasmids carrying GFD1 (pGFD1.1) and DBP5 (pDBP5.3) were
transformed in nab2-20 and the growth phenotypes were examined.
Unfortunately, there was no effect of either gene separately or together on
growth rates at any temperature tested (data not shown). It remains possible

130
that another RNA-dependent helicase is responsible for the dissociation of the
Nab2p-Mex67p-mRNP in the cytoplasm and Gfdlp stimulates the ATPase
activity of this protein as well. For example, DED1 encodes a DEAD-box
helicase with similarity to elF4A and is essential for efficient translation initiation
(Chuang et al., 1997). Importantly, Dedip may have additional, non-essential
functions because ded1 mutants suppress growth defects associated with
splicing defects (prp8-1 strain) and small nuclear RNA transcription defects
(RNAP III mutations) (Jamieson et al., 1991; Thuillier et al., 1995). Dedip has a
predicted molecular weight of 68 kDa and co-purifies with Nab2pT (data not
shown) providing an intriguing possibility that Dedip is the 67 kDa protein
associated with NT2pT, but not nt220pT (Figure 13A). Future studies will be
directed toward the identification of genes that suppress the nab2-20 growth
defect with the hope of demonstrating that Nab2p and Mex67p need to be
dissociated in the cytoplasm in order to recycle back into the nucleus for another
mRNA export cycle.
Limitations
When using a model organism such as Saccharomyces cerevisiae to
study a biological process, it is important to assess the relevance of the results
for higher eukaryotic organisms. Clearly, 3’ end formation is an important
problem for all organisms. However, the cleavage and polyadenylation
machinery has been conserved from yeast to humans with a single glaring
exception, Nab2p. Substantial resources have been expended in the Swanson
Laboratory in order to identify a human protein with sequence and functional

131
similarities to Nab2p, but to no avail. The results presented in this report suggest
PABPN1 is a functional orthologue of Nab2p. Although, PABPN1 does not co¬
purify with abundant hnRNPs in HeLa cells and may not be the only nuclear
poly(A)+ RNA binding protein in metazoan organisms. The situation for PABPN1
and polyadenylation may be similar to that of yeast Pablp in which the human
Nab2p-like protein is inactivated in extracts. In support of this idea, the RGG-
domain from Nab2p is sufficient to import reporter proteins into the nucleus of
two different human cell types. Alternatively, PABPN1 may be responsible for
poly(A)+ tail length regulation in a subset of cell types. Oculopharyngeal
muscular dystrophy (OPMD) is caused by polyalanine expansions in the
PABPN1 protein and specifically affects skeletal muscles, especially those
required for swallowing and eyelid control. Even so, in the absence of a Nab2p
homologue or evidence that PABPN1 is a functional orthologue in yeast cells, we
are left questioning the significance of these findings for understanding poly(A)
tail length control in metazoan organisms.
We have also provided circumstantial evidence that Nab2p may function
in combination with Mex67p to stimulate nuclear export of poly(A)+ RNA.
Unfortunately, evidence directly implicating Nab2p in Mex67p-dependent export
in vivo has not been forthcoming. The nab2-20 mutation, although extremely
useful for in vitro studies, has proved difficult to analyze. Several attempts to
sporulate nab2-20 diploids were unsuccessful. In an attempt to identify
suppressors of the nab2-20 growth defect, cells were transformed with a high-
copy genomic library. Similar numbers of mock-transformed (plasmid only) and

132
library-transformed cells grew-up after five days at 37°. Since nab2-20 was
carried on a low-copy plasmid, we reasoned the mutant allele was reverting to a
wild type allele at a high frequency. Unfortunately, this hypothesis has not been
tested because we have not been able to replace the genomic allele of NAB2
with nab2-20.
Conclusions
In summary, the results presented in this report are suggestive of the
model presented in Figure 21. Nab2p, possibly through interactions with THO
components, Sub2p and/or Nab4p, is recruited to the poly(A) tail during
synthesis. After the addition of ~90 adenosines, Nab2p terminates
polyadenylation, blocks retention of poly(A)+ RNA by the exosome, and facilitates
release from RNAP II. Once the mRNP has been released from the TREX
complex, Mex67p is free to bind to both Nab2p and Yralp. In the absence of
Nab2p, nuclear targeted Pablp blocks nucleolar retention but does not recruit
Mex67p. In this case, Yralp still recruits Mex67p to the mRNA, although with
less efficiency which leads to a partial export block. After recruitment, Mex67p-
Mtr2p associates with Sac3p which transports the mRNA to the nuclear pore.
Once in the cytoplasm, the nuclear poly(A)-binding protein (Nab2p) must be
exchanged for the cytoplasmic poly(A)-binding protein (Pablp) in order for
translation initiation and cytoplasmic turnover to begin. This work has increased
the understanding of poly(A) tail length regulation in yeast and provided a
possible mechanism for coordinating polyadenylation with mRNA export.

APPENDIX
Yeast Strains
Name
Genotype
Source
AH 109
MAT a trp1-901 leu2-3,112 ura3-52 his3-200 gal4A gal80A
LYS2::GAL1uas-GAL1Tata-HIS3 GAL2uas-GAL2Tata-ADE2
URA3::MEL1Uas-MEL 1 TATA-lacZ MEL 1
Clontech
(unpublished)
BMA64
MATa ade2-1 Ieu2-3,112 ura3-1 trp1A his3-11,15 can1-100
(Baudin-Baillieu
et al„ 1997)
DBP5
MATa ade2-101 his3-A200 Ieu2-Al trp1-Al ura3-52 Iys2-801
dbp5::HIS3 pDBP5
Tseng (1998)
dbp5-1
MATa ade2-101 his3-A200 Ieu2-Al trp1-Al ura3-52 Iys2-801
dbp5::HIS3 pdbp5-1
Tseng (1998)
L4717
MATa ade2 can1-100 his3-11,15 Ieu2-3,112 trp1-1 ura3-1
(Hong et al.,
1997)
MEX67
shuffle
MATa ade2 his3 Ieu2 trp1 ura3 mex67::HIS3 pRS316-URA3-
MEX67
Segref (1997)
mex67-5
MATa ade2 his3 Ieu2 trp1 ura3 mex67::HIS3 pUN100-LEU2-
mex67-5
Segref (1997)
PSY714
MATa Ieu2 trp1 ura3 rna1-1
Corbett (1995)
YAS394
MATa pab1::HIS3 RPL46::LEU2 ade2 his3 Ieu2 trp1 ura3
can1
Sachs(1992)
YJA223
MATa leu2A2 his3A200 trp1-189 ura3-52 nab2A::LEU2
TRP::nab2-21
Anderson (1995)
YJA517-1C
MATa \eu2A2 ura3-52 nab2A::LEU2 pGAL::NAB2
Anderson (1995)
YKN105
MATa ade2 can1-100 his3-11,15 Ieu2-3,112 trp1-1 ura3-1
nab2A::HIS3 pPAB1.6
this study
YKN106
MATa ade2 can1-100 his3-11,15 Ieu2-3,112 trp1-1 ura3-1
nab2A::HIS3 pPAB1.7
this study
YKN206
MATa ade2 can1-100 his2-11,15 Ieu2-3,112 trp1-1 ura3-1
TRP1 ::NAB2-TAP
this study
YKN220
MATa ade2 can1-100 his3-11,15 Ieu2-3,112 trp1-1 ura3-1
nab2A::HIS3 pnab2-20
this study
133

134
Yeast Strains
Name
Genotype
Source
YKN230
MATa trp 1-901 leu2-3,112 ura3-52 his3-200 gal4A gal80A
LYS2::GAL1 UAS-GAL1TATA-HIS3 GAL2uas-GAL2tata-ADE2
URA3::MEL1UAs-MEL1TATA-lacZ MEL1 pNAB2.41
this study
YKN231
MAT a trp1-901 leu2-3,112 ura3-52 his3-200 gal4A gal 80A
LYS2::GAL1 uas~GAL1 TATA-HIS3 GAL2uas~GAL2tata-ADE2
URA3::MEL1UAs-MEL1TATA-lacZ MEL1 pNAB2.42
this study
YKN284
MAT a ade2 can1-100 his2-11,15 Ieu2-3,112 trp1-1 ura3-1
pNAB2.48
this study
YKN287
MATa ade2 can1-100 his2-11,15 Ieu2-3,112 trp1-1 ura3-1
pNAB2.54
this study
YKN299
MATa ade2 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1
nab2A::HIS3 pGAL::NAB2
this study
YRH201C
MATa ade2 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1
nab2A::HIS3 pGAL::NAB2
Hector (2000)
YRH204
MATa ade2 can1-100 his3-11,15 Ieu2-3,112 trp1-1 ura3-1
nab2A::HIS3 pPAB1
Hector (2000)
YSD10
MATa TAP::FIP1-TRP1-K1 ade2-1 Ieu2-3,112 ura3-1 trp1A
his3-11,15can1-100
(Dheur et al.,
2003)
Plasmids
Name
Description
pDBP5.3 DBP5 amplified with primers MSS and MSS and sub-cloned into Sp/il-BamHI-cut
YEp13 (Broach et al., 1979)
pGFD1.1 GFD1 amplified with primers MSS1130 and MSS1133 and sub-cloned into
YEp24 (Botstein et al., 1979)
pGST-Nab2- NAB2 amplified with primers MSS1131 and MSS1132 and sub-cloned into
His6 EcoRI-Sa/l-cut pGEX-4T-1 (Smith and Johnson, 1988)
pMEX67.2 Myc-MEX67 fusion gene amplified with primers MSS1606 and MSS1607 and
sub-cloned into Xbal-Ssfl-cut pRD53 (Sikorski and Boeke, 1991)
pMEX67.5 LRR of MEX67 amplified with primers MSS1661 and MSS1662 and sub-cloned
into EcoRI-Sa/l-cut pGEX-4T-1 (Amersham Pharmacia)
pNAB2.41 N-terminus (NT1) of NAB2 amplified with primers MSS1423and MSS1450and
sub-cloned into EcoRI-BamHI-cut pGADT7 (Clonetech)

135
Plasmids
Name
Description
pNAB2.42
N-terminus (NT2) of NAB2 amplified with primers MSS1423 and MSS1444 and
sub-cloned into EcoRI-BamHI-cut pGADT7 (Clonetech)
pNAB2.45
N-terminus (NT2) of nab2-20 amplified from pnab2-20 (Anderson, 1995) with
primers MSS1423 and MSS1444 and sub-cloned into EcoRI-BamHI-cut pGADT7
(Clonetech)
pNAB2.46
5’ UTR and 3’ UTR of NAB2 amplified with primer sets MSS1356/MSS1573 and
MSS1384/MSS1358 and sub-cloned into Sa/l-Psfl-cut and BamHI-Sa/l-cut
pRS315, respectively
pNAB2.47
BamH\-Hind\\\ fragment from pBS1539 (Rigaut et al., 1999) containing the
calmodulin binding peptide, TEV protease, and protein A sequences and sub¬
cloned into pNAB2.46
pNAB2.48
N-terminus of NAB2 amplified with primers MSS1574 and MSS1575 and sub¬
cloned into Psfl-BamHI-cut pNAB2.47
pNAB2.54
N-terminus of nab2-20 amplified from pnab2-20 (Anderson, 1995) with primers
MSS1574 and MSS1575 and sub-cloned into Psfl-BamHI-cut pNAB2.47
pNAB2.58
EcoRI-BamHI fragment from pNAB2.41 containing the NT2 of NAB2 and sub¬
cloned into pET15b (Novagen)
pNAB2.59
EcoRI-BamHI fragment from pNAB2.45 containing the NT2 of nab2-20 and sub¬
cloned into pET15b (Novagen)
pPAB1.3
PAB1 amplified with primers MSS1261 and MSS1259 and sub-cloned intoXhol-
BsfEII-cut pYES2/CT[SPB4] (Invitrogen)
pPAB1.4
Smal-Xbal fragment from pPAB1.3 containing the PAB1-V5 gene fusion was
sub-cloned into pRS315 (Sikorski and Hieter, 1989)
pPAB1.5
3’ UTR of PAB1 amplified with MSS1268 and MSS1288 and sub-cloned into
Xbal-Ssfl-cut pPAB1.4
pPAB1.6
5’ UTR of PAB1 amplified with MSS1287 and MSS1263 and sub-cloned into
Sa/l-Smal-cut pPAB1.5
pPAB1.7
PAB1 amplified with primers MSS1262 and MSS1259 and sub-cloned into Smal-
Sphl-cut pPAB1.6
pPAB1.8
PAB1 amplified with primers MSS1727 and MSS1728 and sub-cloned into
BamHI-Xhol-cut pGEX-4T-1 (Amersham Pharmacia)
pSUB2.1
SUB2 amplified with primers MSS1333 and MSS1334 and sub-cloned into Sph\-
BamHI-cut YEp13 (Broach et al., 1979)
pSUB2.2
SUB2 amplified with primers MSS1333 and MSS1334 and sub-cloned into Sph\-
BamHI-cut YEp24 (Botstein et al., 1979)

136
Plasmids
Name Description
pSUB2.5 SUB2 amplified with primers MSS1729 and MSS1730 and sub-cloned into
BamHI-X/?ol-cut pGEX-4T-1 (Amersham Pharmacia)
pYRA1.16 Exon 1 of YRA1 amplified with primers MSS1539 and MSS1540 and sub-cloned
into BamHI-EcoRI-cut pGEX-4T-1 (Amersham Pharmacia)
pYRA1.17 Exon 2 of YRA1 amplified with primers MSS1541 and MSS1542 and sub-cloned
into EcoRI-Xhol-cut pYRA1.16
DNA Oligonucleotides
Name Sequence Description
MSS811
MSS812
MSS1130
MSS1131
MSS1132
MSS1133
MSS1259
MSS1261
MSS1262
MSS1263
MSS1268
MSS1287
MSS1288
MSS1333
MSS1334
MSS1356
MSS1358
MSS1384
MSS1444
MSS1450
MSS1473
MSS1539
MSS1540
MSS1541
MSS 1542
MSS1573
MSS1574
MSS1575
MSS1606
MSS1607
MSS1661
5’-CCTCCGCAAACCAGTTTTACGCACCAAGAACAAGATACGGA 5’NAB2-TAP
AAT GAACT CCATGGAAAAGAG AAG-3’
5’-ATAGGT GT CTT CCAT CAAAAGGGT CACAGGAACAT G AATTTT 3’NAB2-TAP
CGTTCCGTACGACTCACTATAGGG-3’
5’-GCGGCATGCCCCAGAAACGCATGTATGGACTG-3’ 5’GFD1
5’-GCGGAATT CAT GT CT CAAGAACAGT ACACAGAAAAC-3’ 5’NAB2-H IS6
5’-GCGGTCGACTCAGCCGCTGCTGTGATGATGATGATGATGG 3’NAB2-HIS6
CTGCTGCCGTTCATTTCCGTATCTGTGGCTTG-3'
5’-GCGGGATCCAGT GT GT AT GCT CAATACACAT CACCC-3’ 3’GFD1
5’-GCGGGGT GACCAGCTT GCT CAGTTT GTT GTT CTT GC-3' 3’PAB1-BstEII
5'-GCGCTCGAGCCCGGGATGGCTCCAAAGAAGAAGCGTAAGGT 5’PAB1-NLS
T GAT ATT ACT GAT AAG AC AGCT G AACAATT G-3’
5’-GCGCTCGAGCCCGGGATGGCTCCAAAGACTAAGCGTAAGGT 5’PAB 1 -mtNLS
T GAT ATT ACT GAT AAG AC AGCT G AACAATT G-3’
5’-GCGCCCGGGTTTATTTTTATTGGTTTTTTAGTTTTTTTTGG-3' 3’PAB1-5UTR
5’-GCGTCT AG ATGCT CTATGT AAT C ACCT ACTTCCC-3’ 5’PAB1-3UTR
5’-GCGGTCGACGAGGTCATACTGTATGAAGCCACAAAG-3' 5’PAB1-5UTR
5’-GCGGAGCT CCT GTTACGGTTGCTTTCCCTTGCT C-3’ 3’PAB1-3UTR
5’-GCGGCATGCCATGGAAGATTCGCGTTACCC-3’ 5’SUB2-Sp/?I
5’-GCGGGATCCGTAAGGGTGCTCGTTCTAGATTCC-3’ 3’SUB2-HI
5'-GCGGT CGACGGTT CATT ACAAAATT GAGCCT G-3’ 5’NAB2-Sall
5’-GCGGAGCT CGT GCCTT CCGTTT AGAGT GAGC-3’ 3’NAB2-3UTR
5’-GCGGGATCCTTACTATTAAAATCACGGAACGAAATTC-3’ 5’NAB2-5UTR
5’-GCGGGAT CCTT ACCCT GTTGCAGAGCTT CTAAT GCG-3’ 3’NAB2-NT1
5’GCGGGATCCTTAGTTAACGATCAATAAGACAATATACTCCGC-3’ 3'NAB2-NT2
5-GCGGAATT CT CT CAAGAACAGTACACAGAAAACTT G-3’ 5’NAB2-AD
5-GCGCATAT GT CT GCT AACTT AGAT AAAT CCTT AGAC-3’ 5’YRA1 -E1
5’-GCGG AATT CT CTT AC AGC ATCCTGCTT AAT GTC-3’ 3’YRA1 -E1
5’-GCGG AATT CTTT GCAT CT CAAGT AGGT GGT G-3’ 5’YRA1 -E2
5’-GCGCT CG AGTT ATTT CTTTTCG AAAT AGTCCGC-3’ 3’YRA1 -E2
5’-CGCCT GCAGT GT ACTT CCACTT CCTTAT GAT CAGAG-3’ 3’NAB2-5UTR
5’-GCGCT GCAGT GT ACTT CCACTT CCTTAT GAT CAG AG-3’ 5’NAB2-NT2
5’GCGGGATCCACCCTGTTGCAGAGCTTCTAATGCG-3’ 3’NAB2-NT2
5’-GCGT CT AG AATGGAGGAGCAGAAGCT GAT CT CAGAGG AGGA 5’MEX67-Myc
CCT GAT G AGCGG ATTT CACAAT GTTG-3’
5’-GCGGAGCTCGTGAACGTATGACGATTGAGCTC-3’ 3’MEX67
5’-GCGG AATT CAT GAGCGGATTT CACAAT GTT G-3’ 5’M EX67-LRR

137
DNA Oligonucleotides
Name Sequence Description
MSS1662 5’-GCGGTCGACCTACGGTAATGAGTAAACGGTTTGC-3’ 3’MEX67-LRR
MSS1727 5’-GCGGGAT CCATGGCT GATATT ACT G ATAAGACAGC-3’ 5’PAB1-HI
MSS1728 5-GCGCT CGAGTT AAGCTT GCT CAGTTT GTTCTT GC-3’ 3’PAB1-Xhol
MSS1729 5’-GCGGGATCCACGATGTCACACGAAGGTGAAGAAG-3’ 5’SUB2
MSS1730 5’-GCGCTCGAGTAATTATTCAAATAAGTGGACGGATCAATGC-3’ 3’SUB2

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167
BIOGRAPHICAL SKETCH
Keith Nykamp was born in Holland, Michigan, on May 4, 1974. He is the
oldest of three sons born to Debra Schippers and Randall Nykamp. Keith
attended Hope College in Holland, Michigan, from 1992-1996. During that time,
he pursued a Bachelor of Arts degree in both biochemistry and psychology. After
graduation, Keith worked as a Research Assistant at the Henry Ford Hospital,
Sleep Disorders and Research Center, under the guidance of Drs. Leon
Rosenthal and Thomas Roth. Keith married Dawn DeBoer from Spring Lake
Michigan in May of 1997. In August of 1998, Keith and Dawn moved to
Gainesville for the start of Keith’s graduate career at the University of Florida
College of Medicine, under the auspices of the Interdisciplinary Program in
Biomedical Sciences (IDP). Keith chose to perform his doctorate research under
the guidance of Dr. Maurice Swanson in May of 1999 and completed his
dissertation and graduate work in December of 2003. Keith and Dawn were
blessed with a beautiful and healthy son, Jonas Robert, on September 24, 2002.
Future plans for Keith, Dawn and Jonas include moving to Madison, Wisconsin in
December 2003, to begin a post-doctoral career with Dr. Judith Kimble. Keith will
study germline development using Caenorhabditis elegans as a model organism.

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, iryscppe
and quality, as a dissertation for the degree of Doctor of Phi
itááurice S. Swanson, Chair
Professor of Molecular Genetics
and Microbiology
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctono^ Philosophy^ ^
Alfred Lewin
Professor of Molecular Genetics
and Microbiology
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of DoctOTptPtfilosophy.
Stephen FT Sygrue
Professor of Anatomy and
Cell Biology
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor i
Thomas P. Yang
Professor of Biochemistry
and Molecular Biology
This dissertation was submitted to the Graduate Faculty of thevGollége of
Medicine and to the Graduate School and was accepted as partial fulfillment of
the requirements for the degree of Doctor of Philosophy.
December, 2003
¿entrad-'
DearLCollege of Medicine, ,
Dean, Graduate School




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