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Characterization of a yeast nucleolar protein Nop2p

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Characterization of a yeast nucleolar protein Nop2p
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Hong, Bo
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1 online resource(vii, 137 leaves) : ill. ;

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Nuclear Proteins -- analysis ( mesh )
Cell Nucleolus ( mesh )
Ribosomes -- biosynthesis ( mesh )
Saccharomyces cerevisiae ( mesh )
DNA Mutational Analysis ( mesh )
RNA, Ribosomal -- biosynthesis ( mesh )
RNA Precursors -- biosynthesis ( mesh )
Methylation ( mesh )
RNA Processing, Post-Transcriptional ( mesh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1997.
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Includes bibliographical references (leaves 129-136).
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Typescript.
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Vita.
Statement of Responsibility:
by Bo Hong.

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University of Florida
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University of Florida
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Copyright Bo Hong. 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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879599226 ( OCLC )

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Full Text
CHARACTERIZATION OF A YEAST NUCLEOLAR PROTEIN Nop2p
BY
BO HONG

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

1997




This work is dedicated to my parents




ACKNOWLEDGMENTS

I would especially like to thank my mentor, Dr. John P. Aris, for his guidance and training. I would like to extend my gratitude to my committee members, Dr. William A. Dunn, Dr. Alfred S. Lewin, and Dr. Maurice S. Swanson, for their encouragement and support. I would also like to thank the faculty and staff at the Department of Anatomy and Cell Biology for providing a stimulating intellectual environment for my graduate training. Dr. J. Scott Brockenbrough deserves my special thanks for his assistance and friendship. Most important, I would like to thank my wife Liu for her understanding and support.




TABLE OF CONTENTS
page
ACKNOWLEDGMENT ................................................................... iii
A B STR A C T IO N. ...................... ....................................................vi
INTRODUCTION ...............................................................
The Nucleolus and Ribosome Synthesis ...................................... 1
Processing of Pre-rRNA ........................................................ 3
Modification of rRNAs and Ribosomal Proteins .............................. 6
Functions of the Nucleolar Proteins ............................................ 9
Functions of SnoRNAs ........................................................... 17
Interaction of Nucleolar Proteins and SnoRNAs .............................. 18
MATERIALS AND METHODS ......................................................... 20
Strains, Media, and Microbiological Methods ................................. 20
DNA Methods ............................................ 21
Construction of GAL-NOP2 Strain ............................................ 23
Cell Growth Curve and Viability Test .......................................... 31
Isolation of nop2 Conditionally Lethal Alleles ................................ 32
Mapping of the Mutations in the Six nop2 Alleles ............................ 33
SDS-PAGE and Immunoblotting ............................................... 34
Measurement of Ribosomes and Polysomes ................................... 34
Analysis of Pre-rRNA Processing by Pulse-Chase Labeling ................ 35
Northern Analysis of rRNAs .................................................... 35
Primer Extension .............................................................. 36
In vitro Methylation Analysis .................................................... 36
RNase Protection Assay .......................................................... 37
Immunoprecipitation Analysis ................................................... 38
Screening for High Copy Suppressors ......................................... 40
R E SU L T S ................................................................................... 50
D epletion of N op2p ............................................................... 50
Mutational Analysis of Nop2p ................................................... 69
Nop2p and Methylation ........................................................... 96
Interaction of Nop2p with other Macromolecules .............................. 109




DISCUSSION ................................................................ 117
Nop2p is Essential for Cell Viability and Growth ....................... 117
Nop2p is Required for Ribosome Synthesis............................ 118
Interaction of Nop2p with Other Macromolecules ....................... 122
The Mechanism by Which Nop2p Functions ........................... 123
REFERENCES ............................................................... 129
BIOGRAPHICAL SKETCH .................................................. 137




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 CHARACTERIZATION OF A YEAST
NUCLEOLAR PROTEIN Nop2p
By
Bo Hong
August 1997
Chairman: John P. Aris, Ph.D.
Major Department: Anatomy and Cell Biology
The main function of the nucleolus is the synthesis of ribosomes, which involves a series of complicated events, including processing and modification of pre-rRNA. It has been proposed that nucleolar proteins play important roles in these events. However, only a few nucleolar proteins have been identified so far, and the detailed molecular mechanisms related to ribosome synthesis remain elusive. The yeast nucleolar protein Nop2p is essential for cell viability and growth in Saccharomyces cerevisiae. In order to dissect its function, conditionally lethal strains were constructed either by placing the open reading frame under the control of a repressible promoter, GALIO, or by mutagenizing the gene. Depletion of, and mutation in, Nop2p caused a significant reduction in the levels of the 25S rRNA and the 60S ribosomal subunit. Pulse-chase analysis of the processing of pre-rRNA in these conditionally lethal strains revealed that the processing was significantly delayed with the accumulation of 35S, 32S and 27S pre-rRNAs and with a significant reduction in the formation of 25S and 5.8S rRNAs. Northern blotting analysis indicated that both 27SA and 27SB pre-rRNAs accumulated during Nop2p depletion. Since protein alignment




analysis suggests that the protein may have methylase activity, the role of Nop2p in methylation of rRNAs was investigated by RNase protection assay. Depletion of Nop2p caused a statistically significant reduction in methylation at a conserved site in 27S prerRNA. Mutational analysis of the protein revealed that the multiple missense mutations in the gene were responsible for the temperature-sensitive or formamide-sensitive phenotypes. The mutations in the six nop2 alleles caused a defect in the processing of pre-rRNA to 25S rRNA. The 27S pre-rRNA, which accumulated in the other four nop2 alleles, was rapidly degraded in nop2-4 and nop2-5 alleles. These findings clearly indicate that Nop2p is required for the synthesis of 60S ribosomal subunit and processing of 27S pre-rRNA to 25S and 5.8S rRNAs. This study also helps elucidate the molecular mechanisms related to ribosome synthesis.




INTRODUCTION

The Nucleolus and Ribosome Synthesis
The nucleolus is a membrane free organelle within the nucleus. Morphologically, three substructures can be recognized in the interphase nucleoli of higher eukaryotic cells: fibrillar centers (FCs), dense fibrillar component (DFC) and granular component (GC). (Warner, 1990; Scheer et al., 1993; Scheer and Weisenberger, 1994). Since FCs contain rDNA and the enzymes essential for its transcription (RNA polymerase I, DNA topoisomerase I and transcription factors), it is believed that the primary rRNA transcript is generated in FCs. Fibrillarin, a nucleolar protein, was found to be localized in this substructure. Since fibrillarin is involved in pre-rRNA processing, DFC is likely the site of early steps in pre-rRNA processing. The late events of maturation of ribosome precursor particles occur in GC. The yeast nucleolus appears as a single, crescent-shaped region occupying more than 30% of the nucleus (Woolford, 1991). Although there are no obviously distinct substructures in the yeast nucleolus, ribosome synthesis is also the main function of yeast nucleolus (Warner, 1989; Melese and Xue, 1995).
The yeast ribosome is a large complex of proteins and RNAs, which consists of
40S and 60S ribosomal subunits. The 40S subunit contains about 32 different proteins and an 18S rRNA, while the 60S subunit contains about 45 proteins and three rRNAs: 5S,
5.8S and 25S (28S in higher eukaryotes) rRNAs (Woolford, 1991). Since the ribosome is a protein-synthesizing machine, synthesis of the ribosome itself is essential for cell growth and maintenance. The nucleolus is the center for ribosome synthesis (Table 1). In the nucleolus, repeated rDNA genes are transcribed by RNA polymerase I into a single large




Table 1. Main Events in Ribosome Synthesis Event Location Comments

rDNA transcription 5S rDNA transcription

Pre-rRNA processing
Modification of rRNAs
Transcription of ribosomal protein (r-protein) genes Processing of mRNAs of r-proteins
Translation of r-proteins Modification of r-proteins Import of r-proteins

Nucleolus Nucleolus

Nucleolus (except 20S to 18S rRNA in the cytoplasm)

Nucleolus
Nucleus
Nucleus Cytoplasm Cytoplasm or nucleolus? Cytoplasm to nucleolus

Transcribed by RNA polymerase I into a large single precursor containing 18S, 5.8S and 25S rRNA sequences.
Transcribed by RNA polymerase II; no processing or modification has been found.
Involving a series of cleavages; simultaneous with the modification and assembly.
Including base methylation, ribose methylation, and pseudouridylation during the transcription.
RNA polymerase II.
Including capping, splicing and polyadenylation.
Including methylation, phosphorylation and acetylation.
Involving nuclear localization signal (NLS) or "piggy-back import" mechanism.

Involving protein-protein and protein-RNA interaction.

Export of ribosome subunits

Nucleolus to cytoplasm

Assembly

Nucleolus




35S (45S in higher eukaryotes) precursor, which is then modified and cleaved into three mature rRNAs: 18S, 5.8S and 25S rRNAs. 5S rRNA is generated by RNA polymerase III. Coupled with this maturation process, ribosomal proteins, imported from the cytoplasm, are assembled with pre-rRNAs and 5S rRNA to form functional 40S and 60S ribosomal subunits. The ribosomal subunits are then exported to the cytoplasm for protein synthesis (Warner, 1989; Woolford and Warner, 1991; Sollner-Webb et al., 1996).
Processing of Pre-rRNA
The single, large pre-rRNA contains 18S, 5.8S and 25S rRNA sequences, which are flanked by 5' and 3' external transcribed spacer (ETS), and separated by internal transcribed spacer (ITS) 1 and 2 (Figure 1). Release of the rRNAs from the precursor is, therefore, essential for ribosome synthesis. Processing of pre-rRNA into mature rRNAs involves multiple endonucleolytic and exonucleolytic cleavages. Earlier studies of prerRNA processing in unicellular organisms (Saccharomyces cerevisiae), plants (Phaseolus vulgaris), insects (Drosophila), amphibia (Xenopus laevis) and several mammals suggest that a common processing pathway is generally followed (Crouch, 1984; Hadjiolov, 1985; Tollervey, 1987; Gerbi et al., 1990; Eichler and Craig, 1994; Sollner-Webb et al., 1996).
In yeast, the processing starts at 5' ETS (Figure 1). Cleavage at A0 generates a 33S intermediate, which is then converted to 32S with the processing at A1 site. These early processing steps generate the 5' end of 18S rRNA. Cleavage at A2 within ITS 1 separates the processing pathway into two halves. One is conversion of 20S into 18S rRNA, which occurs in the cytoplasm. Another is to form 5.8S and 25S rRNAs. When the processing of ITS I and 3' ETS is completed, 27SA becomes 27SB. 27SB is the common precursor for 5.8S and 25S rRNAs. Cleavage at C2 site within ITS2 generates two intermediates. One contains ITS2 and 25S rRNA sequence. With the subsequent processing at C1, the mature 25S rRNA is formed. Another is 7S intermediate, the direct precursor for 5.8S




Figure 1. The pre-rRNA processing pathway in Saccharomyces cerevisiae. The rDNA is transcribed into a single 35S pre-rRNA, which contains the 5' external transcribed spacer (ETS), the sequences of 18S, 5.8S and 25S rRNAs separated by the internal transcribed spacer (ITS) 1 and 2, and 3' ETS. To release the rRNA sequences, the 35S pre-rRNA undergoes a series of endonucleolytic and exonucleolytic cleavage steps as indicated. The cleavage at A2 site in ITS I separates processing into the two pathways leading to the rRNAs of the large and small ribosomal subunits, respectively. The processing of 20S to 18S rRNA occurs in the cytoplasm. The cleavage of 27SB at C2 site in ITS2 generates the precursors for 5.8S and 25S rRNAs, respectively. There are two forms of 5.8S rRNA: the major short form 5.8SS, and the minor large form 5.8SL (with the extra 7-8 nucleotides at the 5' end, Henry et al., 1994).




5' ETS ITS 1 BL ITS 2
AO Al D A2 A3 BlS E C2 C1
35S

Processing AO Cleavage Al
32S
SCleavage A2

20 S ISCleavage D 18s

27SBL
7 y Proces
7SL Exonuc
5.8SL 25S L

Processing BIL & B2

sing Cl & C2 lease C2 > E

I- 27S A2

Cleavage A3 Processing B2 S27SA3 Exonuclease A3 > B1S 27SBs

Cleavage c2 7SS
Exonuclease
C2 > E Cleavage ci
5.8SS

25S

3' ETS
B2

ii

----I




rRNA. With exonucleolytic "chewing" from C2 to E site, 7S is converted to 5.8S rRNA (Tollervey, 1987). A recent study revealed that there are two versions of 5.8S rRNA in the wild type cells. The larger 5.8S (5.8SL) containing several extra nucleotides at 5' end is a minor version (Henry et al., 1994).
Modification of rRNAs and Ribosomal Proteins
Methylation of rRNAs
All eukaryotic rRNAs are modified by methylation, though the number of the
methylations varies among different organisms. There are a total of 116 methylations in human rRNAs, while only 65 methylations in yeast rRNAs (Maden et al., 1995). Nevertheless, from yeast to human, a great majority (80-90%) of the methylations are 2'O-methylations. No consensus sequence for rRNA methylation has been found. However, almost all eukaryotic rRNA methylations are in the conserved core regions of rRNA: the regions with main secondary structure features conserved from archaea to bacteria to eukaryotes (Maden, 1990; Maden, 1996). In yeast, most methyl groups are added soon after transcription at the level of 35S and 32S precursors (early methylation). Very few are added at the level of the late processing intermediates at the level of 27S and 20S intermediates (late methylation). Among the methylations identified in the 25S rRNA sequence, only four were found to be added at the level of 27S level: two copies of mU (base methylations) and UmGmTUC2922 (2'-O-methylations) (Brand et al., 1977).
The function of the methylation remains elusive. Since methyl groups add
hydrophobic patches to rRNA and since all methylations are in the conserved core regions, the modification may facilitate rRNA folding or rRNA-protein interactions, and thus may be involved in ribosome synthesis. Some earlier studies found that the methylation of rRNAs was required for pre-rRNA processing and maturation of ribosomes. In cultured




mammalian cells, methionine deprivation or treatment with ethionine, a chemical inhibitor of methyl transfer, caused a defect in ribosome maturation and pre-rRNA processing (Swann et al., 1975; Caboche and Bachellerie, 1977; Wolf and Schlessinger, 1977).
The methylation of rRNA may also be involved in the ribosome function. A higher concentration of methyl groups was found in and around the peptidyl transfer region of the large subunit rRNA, and several modifications in 18S rRNA were identified on the platform of the small ribosomal subunit, where mRNA and tRNA binding occurs (Maden, 1995). Demethylation of rRNAs was found to be related to the reduced protein synthesis (Wolf and Schlessinger, 1977). Recent studies of Dim lp (a yeast 18S rRNA methylase) and a yeast temperature-sensitive nopl mutant suggest that the ribosome containing the demethylated rRNAs was defective in its function. However, the demethylated pre-rRNA was still processed and methyl deficient ribosome could still be formed (Tollervey et al., 1993; Lafontaine et al., 1995).
These findings suggest that rRNA methylation may be required for ribosome
function. Nevertheless, the relationship between methylation and pre-rRNA processing and ribosome maturation has not been clearly understood.
Pseudouridylation of rRNAs
Eukaryotic rRNAs are also modified by pseudouridylation. The ratio of
pseudouridylation to 2'-O-methylations is close to 1:1 in yeast (47:55), amphibian (98:98) and human (95:105) rRNAs (Maden et al., 1995), suggesting that the two types of modifications may be interdependent. Since pseudouridine has more versatile hydrogenbonding properties than uridine, it was speculated that pseudouridine contributes to the recognition events crucial to ribosomal function. However, the exact function of this modification has not been understood.




Modification of Ribosomal Proteins
Ribosomal proteins were found to be modified by methylation, phosphorylation or acetylation (Raue and Planta, 1991; Woolford and Warner, 1991). Several yeast ribosomal proteins belonging to the small subunit (S31 and S2) and to the large subunit (L3, L15, LA1, and L42) were found to be methylated (Cannon et al., 1977; Kruiswijk et al., 1978). Since addition of a methyl group increases the local hydrophobicity, it is possible that methylation of ribosomal proteins may be related to protein-protein association during the assembly. The study of methylation of the ribosomal protein L3 in E.coli supports this possibility. The only modification in L3 is N5-methylglutamine, which is modified by the prmB protein. The E.coli mutant lacking this methylase activity displayed a cold-sensitive phenotype. At 220C, the mutant cells synthesize abnormal ribosomal particles that are very unstable and quickly degraded (Lhoest and Colson, 1977; Muranova et al., 1978; Lhoest and Colson, 1981). However, in eukaryotic cells, such protein methylases have not been identified. The role of methylation of ribosomal proteins in ribosome synthesis is not known.
The function of phosphorylation and acetylation of ribosomal proteins is not known. The lack of acetylation (such as in rp39, rp52, rp56 and rp59) or the lack of phosphorylation (such as in S 10) did not affect cell growth, suggesting that these modifications may not function in ribosome synthesis (Johnson and Warner, 1987; Woolford and Warner, 1991; Wachtler and Stahl, 1993).




Functions of Nucleolar Proteins
Pre-rRNA Processing
Non-ribosomal proteins in the nucleolus are thought to play important roles in the
synthesis of ribosomes. The number of these nucleolar proteins is believed to be very large (Hernandez-Verdun, 1991). A number of the nucleolar proteins required for rDNA transcription have been identified (Reeder, 1990). However, the functions of the nucleolar proteins in the other events of ribosome synthesis have just recently become understood. Although the general outline for pre-rRNA processing (Figure 1) has been known for years, the enzymes and regulatory factors required for the processing have been largely unknown until recently. Several nucleolar proteins, such as fibrillarin, B23 and nucleolin, were described in high eukaryotic cells years ago (Michalik et al., 1981; Ochs et al., 1985; Caizergues et al., 1987). However, their functions remain unclear, mainly because of technical difficulties in the study of high eukaryotic cells. The yeast system is easily accessible to powerful genetic and molecular biology techniques. Recent studies in the yeast model have generated a large amount of knowledge about the roles of the nucleolar proteins in pre-rRNA processing (Figure 2).
Fibrillarin, localized in the DFC of the nucleolus, was first found in Physarum and later identified in mammalian cells (Christensen et al., 1977; Ochs et al., 1985). Since its yeast homologue, the Nopl protein (Noplp), was identified (Aris and Blobel, 1991), the protein function has been extensively studied. Human or Xenopus fibrillarin can complement a yeast nopl mutant (Jansen et al., 1991), suggesting that the protein function is evolutionarily conserved. By genetic manipulation, NOP1 was placed under the control of a repressible promoter. Depletion of this protein caused a progressive impairment of all pre-rRNA processing steps, particularly the pathway leading to 18S rRNA (Tollervey et al., 1991). The finding suggests that Noplp is required for the processing in 5' ETS and




Figure 2. Yeast nucleolar proteins implicated in the processing of pre-rRNA. The nucleolar proteins listed in the boxes are required for the processing of pre-rRNA. Depletion of, or mutation in, the proteins separately caused a reduction in the production of rRNAs. Some of the proteins are involved in the processing in more than one spacer region. The arrows indicate the spacer regions of 35S pre-rRNA requiring the nucleolar proteins for processing. *: Nop3p is localized in the nucleus.




5' ETS

35S 18S 5.8S

25S

Noplp Nop3p* Nop4p Dbp3p Drslp Spb4p Rrplp ? Ratlp RNaseMRP Xmlp

Rntlp (RNase III)

i i i l !

ITS 1

ITS 2

3' ETS




ITS 1. Further study of the temperature sensitive (ts) mutations in NOP1 gene showed that the five nopl ts alleles affected ribosome synthesis at different steps, including pre-rRNA processing, pre-rRNA modification and ribosome assembly (Tollervey et al., 1993). The Sofl protein was isolated by screening for the extragenic suppressors of the ts phenotype caused by incomplete complementation of a nopl disruption by human fibrillarin. Soflp was found to be associated with Noplp, and depletion of it caused a defect in 18S rRNA production (Jansen et al., 1993), suggesting that Soflp is required for processing in the 5' ETS and/or ITS 1. Another nucleolar protein required for 18S rRNA formation is Garlp, which shares a glycine, arginine-rich domain (GAR domain) with Noplp (Girard et al., 1992). Nop3p, also sharing a GAR domain with Noplp, is required for the conversion of 27SB pre-rRNA to 25S rRNA, and 20S pre-rRNA to 18S rRNA (Russell and Tollervey, 1992). Nop77p was isolated by screening for genes synthetically lethal with nopl-5 ts allele (Berges et al., 1994). The same protein was also identified by another lab at the same time and termed Nop4p (Sun and Woolford, 1994). Nop77p/Nop4p is required for the conversion of 27SA to 27S B pre-rRNA, since no 27SB pre-rRNA was formed in the cells depleted of the protein. This suggests that the protein is required for the processing in 3' ITS 1. All these findings indicate that fibrillarin (Noplp) is the center of more than one protein-protein and protein-snoRNAs (see below) complex, which mainly functions in the processing of 5' ETS and ITS 1.
Several putative ATP-dependent RNA helicases in yeast have been characterized. These proteins contain the evolutionarily conserved DEAD-box. The DRS1 gene was isolated by complementation of cold-sensitive phenotype of drsl. In the drsl mutant, processing of 27S pre-rRNA to 25S rRNA was impaired (Ripmaster et al., 1992). Another yeast putative RNA helicase is Spb4p, which is also involved in the processing of 27S pre-rRNA to 25S rRNA (Sachs and Davis, 1990). However, it is not clear if Drslp and Spb4p are involved at the step of 27SA to 27SB pre-rRNA, or at the step of 27SB to 25S rRNA. Recently, another putative RNA helicase, Dbp3p, was isolated. This protein




was found to be required for processing at A3 site in ITS 1. The gene is not essential. However, disruption of it caused a delay in the processing of 27SA to 27SB pre-rRNA, and thus a significant reduction in the formation of 25S rRNA (Weaver et al., 1997). The mechanism by which these putative RNA helicases are involved in pre-rRNA processing is not clear. It was speculated that RNA helicase may modulate the secondary structure of pre-rRNA for recruiting the RNase MRP complex (see below) to A3 site (Weaver et al., 1997).
Several other proteins were found to be involved in processing ITS 1. In rrpl ts
mutant, 27S pre-rRNA was rapidly degraded and the formation of 25S rRNA was reduced (Fabian and Hopper, 1987). It is not clear if 27SB pre-rRNA was formed or not. RNase MRP, an endoribonuclease, is required for processing at A3 site in ITS 1. Mutation in its protein component (ts popl-1) or in its RNA component (rrp2-1) caused a defect in the cleavage at the A3 site (Lygerou et al., 1994; Morrissey and Tollervey, 1995). Xrnmlp and Ratlp were found to have 5' to 3' exonuclease activity. Mutation in RAT1 gene caused a reduction in the formation of the short form of yeast 5.8S rRNA (Amberg et al., 1992). Mutation in XRN1 gene resulted in the accumulation of the excised 5' region of ITS 1, between the 3 end of 18S rRNA and A2 site (Stevens et al., 1991). Ratlp and Xrnlp were found to digest from site A3 to B 1 (s) to form the 5' end of 27SB (s) pre-rRNA (Henry et al., 1994).
Yeast RNT1, a homologue of bacterial RNase III, was found to encode a doublestrand endoribonuclease, which is required for processing both the 5' and the 3' ETS. This endonuclease activity was reproduced in vitro in the absence of snoRNA, suggesting that snoRNA had no catalytic role in this situation (Elela et al., 1996). Nsrlp, a yeast homologue of nucleolin, is required to form 20S pre-rRNA, a direct precursor for 18S rRNA. The cells lacking NSR1 were viable but had a severe growth defect and an near absence of 20S pre-rRNA (Lee et al., 1992).




Since pre-rRNA processing involves multiple orderly cleavage steps (Figure 1), it is not surprising that so many nucleolar proteins are required for its processing. So far, no yeast nucleolar protein has been clearly shown to be required for ITS2 processing. The detailed mechanisms for the other processing steps have still not been completely understood. Therefore, much more remains to be learned about the functions of the nucleolar proteins in pre-rRNA processing.
Modification of rRNAs
It has been known for many years that all eukaryotic rRNAs are modified by methylation (Maden, 1990). However, only recently have several putative eukaryotic rRNA methylases and the regulatory factors been characterized. Noplp was found to be required for rRNA methylation (Tollervey et al., 1993). Since Noplp is associated with a large number of the nucleolar proteins and snoRNAs (see below) and since it was found to be involved in every major step of ribosome synthesis (Tollervey et al., 1993), it is most possible the Nop I p is a multi-functional snoRNP protein. Human proliferation-associated nucleolar protein p 120 was found to contain a sequence conforming to an S-adenosylmethionine (SAM) binding domain, which is present in a variety of the known methylases (Koonin, 1994). However, it has not been shown that p 120 possesses rRNA methylase activity. Segal and Eichler identified and partially purified a novel nucleolar methylase from the crude mouse nucleolar extracts. This protein was found to methylate the 28S rRNA transcript at a unique site in vitro (Segal and Eichler, 1991). However, the gene has not been isolated.
Recently, two yeast rRNA methylases have been identified. Pet56 protein was found to be required for ribose methylation at a universally conserved nucleotide in the peptidyl transferase center of the mitochondrial large ribosomal RNA (21S rRNA). This was confirmed by the result from the in vitro methylation reaction. The reduction in this




protein function caused a defect in the formation of large subunit of mitochondrial ribosome (Sirum-Connolly and Mason, 1993), which suggests that the rRNA methylation may be required for ribosome assembly or stability. Yeast Diml protein was cloned by complementation of the kasugamycin sensitivity of the E.coli ksg A mutant (Lafontaine et al., 1994). KsgA is a 16S rRNA dimethylase in E.coli (van Buul and van Knippenberg, 1985). The study of Dimlp function showed that this protein also had dimethylase activity in yeast. This protein was found to be required for the methylation at a specific site on 18S rRNA. However, such methylation was not required for the formation of 18S rRNA, though Dimlp itself was essential for cell viability (Lafontaine et al., 1995).
Considering that all eukaryotic rRNA are methylated at a large number of sites, more rRNA methylases probably remain to be identified, and the significance of rRNA methylation in ribosome synthesis remains to be studied.
Modification of Proteins
Ribosomal proteins are covalently modified (Raue and Planta, 1991; Woolford and Warner, 1991). However, it is not known if such modification occurs in the cytoplasm and/or the nucleolus. Since all of the ribosomal proteins are imported to the nucleolus to assemble with rRNAs into ribosome subunits, it is possible that the nucleolar proteins may modify some of the ribosomal proteins to facilitate the assembly.
Some nucleolar proteins, such as nucleolin, fibrillarin, and Ssblp, were found to be methylated (Lischwe, 1990). All of these nucleolar proteins contain the glycine, arginine-rich domain (GAR domain) interspersed with phenylalanine, which is thought to be recognized by a group of protein methylases (Lischwe, 1990). The methyl groups were found to be added to the arginine residues as NG, NG-dimethylarginine (Lischwe et al., 1982; Lischwe et al., 1985). The exact function of the arginine methylation is not known. Since the GAR domain was found to have a high affinity for nucleic acid (Cobianchi et al.,




1988), it was believed that such modification is related to nucleic acid binding. Since the methyl group increases the local hydrophobicity, a role of the modification in proteinprotein interaction is also possible (Lischwe, 1990). However, the methylases required for the modification have not been identified. These nucleolar proteins are mainly localized in the nucleolus (Lischwe et al., 1985; Lapeyre et al., 1987; Clark et al., 1990), Therefore, it is possible that other nucleolar proteins with protein methylase activity may modify these proteins.
Assembly of Ribosomes
Ribosome assembly involves association of approximately 70-80 ribosomal
proteins and 4 rRNAs through protein-protein and protein-rRNA interactions. In E. coli, ribosomal subunits can be successfully assembled in vitro with only rRNA and ribosomal proteins (Nomura, 1973). However, such in vitro assembly has not been successful for eukaryotic ribosomes, suggesting that some cofactors are required for proper assembly. In vivo labeling in yeast showed that ribosomal precursor particles contained not only ribosomal proteins, but also a number of non-ribosomal proteins (Warner, 1971), suggesting that nucleolar proteins may participate in the assembly process.
Nucleolin and B23 in higher eukaryotes were found to be associated with preribosomal particles in the granular component of the nucleolus (Spector et al., 1984; Biggiogera et al., 1989), where pre-ribosomal subunits occur. Since both proteins contain acidic domains (Spector et al., 1984; Lapeyre et al., 1987), it was speculated that the proteins may function by facilitating assembly of the many basic ribosomal proteins with rRNAs (Woolford, 1991). Several other nucleolar proteins also contain an acidic domain, including Noppl40, Nsrlp, Drslp, and Nop77p/Nop4p (Melese and Xue, 1995). It was believed that the acidic domain in these proteins may interact with the highly basic NLS or NLS-like sequences in many ribosomal proteins and thus to facilitate ribosome assembly




(Xue and Melese, 1994; Melese and Xue, 1995). The glycine, arginine-rich domain (GAR domain) is present in many nucleolar proteins, including fibrillarin (Noplp), Nsrlp, Ssblp, nucleolin, Garlp, and Nop3p (Schimmang et al., 1989; Lee et al., 1991; Jong et al., 1987; Lapeyre et al., 1987; Girard et al., 1992; Russell and Tollervey, 1992). It is possible that GAR domain in these proteins may promote proper assembly of ribosome by interacting with some acidic ribosomal proteins, such as yeast AO, Al, L44, L44' and L45 (Woolford and Warner, 1991).
Since ribosome assembly involves interaction among ribosomal proteins and
rRNAs, modification of rRNA by the nucleolar proteins may play a role in assembly. One piece of evidence is from the study of a yeast ts nopl.3 mutant (Tollervey et al., 1993). In this mutant, the methylation of pre-rRNA was strongly inhibited at 360C, but the demethylated pre-rRNA was still processed. However, the lethal phenotype caused by this allele at 360C suggests that the synthesized ribosomes were non-functional.
Functions of SnoRNAs
Small nucleolar RNAs (snoRNAs) have been identified in a variety of eukaryotic cells (Fournier and Maxwell, 1993). The number of snoRNAs is very large. In yeast, more than 30 snoRNAs have been isolated (Balakin et al., 1996). Some snoRNAs have been shown to be required for pre-rRNA processing. Depletion of either U3, U 14, or snR30 caused a severe defect in the formation of 18S rRNA (Li et al., 1990; Hughes and Ares, 1991; Morrissey and Tollervey, 1993), suggesting that these snoRNAs function in the processing of 5' ETS and/or ITSI1 (Fournier and Maxwell, 1993). SnR 10, though not essential, participates in the processing of 35S pre-rRNA (Tollervey, 1987). However, other snoRNAs have not been found to be essential for cell viability and pre-rRNA processing (Sollner-Webb et al., 1996).




Recently, it has been found that a family of snoRNAs are required for the site selection of rRNA 2'-O-methylation (Maden, 1996; Tollervey, 1996). There are two important features about these snoRNA. First, they contain two evolutionarily conserved motifs, boxes C and D, which are believed to be protein binding sites. Second, the snoRNAs exhibit long (14-17 nucleotide) regions of perfect complementarity to pre-rRNA (Maden, 1996; Tollervey, 1996). Mutations altering the sequence of the snoRNA or the spacing between box D and the region of base paring is sufficient to change the specificity of methylation (Cavaille et al., 1996; Kiss-Laszlo et al., 1996). These studies suggest that the 2'-O-methyltransferase may consist of a protein component, which catalyzes transfer of a methyl group, and an snoRNA component, which selects the modification site.
Interaction of Nucleolar Proteins and SnoRNAs
Several pieces of evidence indicate that nucleolar proteins and snoRNAs may
functionally and physically interact. First, depletion of Noplp, Garlp, Soflp and Nsrlp cause phenotypes that are strikingly similar to phenotypes caused by depletion of U3, U14, and snR30, which suggests that these proteins and RNAs function at the same or similar steps of pre-rRNA processing (Li et al., 1990; Hughes and Ares, 1991; Tollervey et al., 1991; Girard et al., 1992; Lee et al., 1992; Jansen et al., 1993; Morrissey and Tollervey, 1993). Second, immunoprecipitation studies indicate that the nucleolar proteins are physically associated with snoRNAs. At least 11 yeast snoRNAs can be precipitated by antibodies to Noplp (Schimmang et al., 1989). Garlp and Soflp were found to be associated with snRIO and snR30 (Girard et al., 1992), and with U3 (Jansen et al., 1993), respectively. Finally, a majority of the nucleolar proteins identified so far ( such as Noplp, Nop3p, Garlp, Nsrlp, Ssblp, fibrillarin and nucleolin) contain the consensus motifs of RNA recognition motif (RRM) or/and GAR domain (Ochs et al., 1985; Jong et al., 1987; Lapeyre et al., 1987; Schimmang et al., 1989; Lee et al., 1991; Girard et al., 1992; Russell




and Tollervey, 1992). The RRM consists of an amino acid sequence of ~80 residues conserved among the members of a family of RNA-binding proteins (Kenan et al., 1991). The aromatic and basic residues in the RRM have been implicated in nucleic acid interaction (Kenan et al., 1991). The GAR domain consists of repeating units of RGGFRGG (Russell and Tollervey, 1992). Study of the GAR domain of vertebrate nucleolin suggests that the domain is involved in non-specific protein-RNA association (Ghisolfi et al., 1992). On the other hand, all fibrillarin (Noplp)-associated snoRNAs contain two consensus sequences, boxes C and D, which may be the binding sites for common proteins (SollnerWebb et al., 1996). Another family of snoRNAs have consensus ACA triplet positioned 3 nucleotides before the 3' end of the RNA. All members of the yeast ACA family are associated with Garlp (Balakin et al., 1996). Based on the above evidence, it was proposed that nucleolar proteins are associated with snoRNAs to form snoRNP complex, which may be involved in the processing and modification of pre-rRNA, ribosome assembly and transport (Fournier and Maxwell, 1993).
Nop2p is a yeast homologue of human nucleolar protein p120, which is a marker for cell proliferation (de Beus et al., 1994; Fonagy et al., 1993). Nop2p is different from most of the known nucleolar proteins in that it does not contain either RRM or GAR domain (de Beus et al., 1994). However, like p 120, Nop2p contains a putative Sadenosyl-methionine (SAM) binding domain, which is conserved among methylases. In this study, by genetic manipulation, the conditionally lethal strains were constructed and a deficit in Nop2p function was investigated at the restrictive conditions. The relationship between Nop2p and methylation was analyzed. By using immunological and genetic approaches, possible functional interaction between Nop2p and other macromolecules was explored.




METHODS AND MATERIALS

Strains, Media, and Microbiological Methods
Saccharomyces cerevisiae strains used in this study are listed in Table 2. Yeast
cells were grown at 300C or as indicated. The complex media were either YPD (1% yeast extract, 2% peptone, and 2% dextrose) or YPGal (1% yeast extract, 2% peptone, and 2% galactose). The synthetic minimal media were either SD (0.67% yeast nitrogen base and 2% dextrose) or SGal (0.67% yeast nitrogen base and 2% galactose), supplemented with appropriate nutrients (Sherman, 1991). Methionine was always omitted from the synthetic media in this study. For [3H]uracil labeling experiment, the concentration of uracil in the synthetic media was reduced to one third the normal amount (Sherman, 1991). For plasmid shuffling, 1 mg/ml of 5-fluoroorotic acid (5-FOA) (PCR Incorporated) was added to either SD or SGal medium (Boeke et al., 1987). For screening for formamide-sensitive or ethanol-sensitive strains, 3% formamide (Aguilera, 1994) or 6% ethanol (Aguilera and Benitez, 1986) was added into SD medium, respectively. YPD containing low phosphorus (LP-YPD) was prepared as described previously (Reid and Schatz, 1982). Briefly, YP (1% yeast extract, 2% peptone) was treated with 10 mM MgSO4 and 1 % NH4OH at room temperature (RT, ~250C) for 30 minutes, and filtered twice through Whatman 3MM paper before adding 2% dextrose. For screening for thermosensitive strains, yeast cells were replica plated and grown at permissive temperature (250C) and non-permissive temperature (140C or 370C).
Yeast genetic manipulation was done according to previously described methods (Guthrie and Fink, 1991) with minor changes. Yeast cells were first grown at 300C on




selective SD plates for 2-4 days. A small dab of cells was patched onto the pre-sporulation plate (0.8% yeast extract, 0.3% peptone, 10% dextrose, and 1.5% agar.) and grown at 300C for -24 hours. Cells from pre-sporulation were then made a patch onto the sporulation plate (1% KOAc, 0.1% yeast extract, 0.05% dextrose, 1.5% agar, and with one fourth the normal amount of appropriate nutrients). The spore wall of asci was digested with 6% glusulase (DuPont) at room temperature for 6 minutes. The digested asci were then spread onto a YPD plate, and spores were separated by micromanipulation. The spores were germinated and grown on YPD plates.
DNA Methods
All of the plasmids used in this study (Table 3) were maintained and amplified in E. coli DH5a in LB medium (0.5% Bacto-yeast extract, 1% Bacto-tryptone, 0.5% NaC1, pH 7.5, and 1.5% agar for plates only) plus 100 gg/ml ampicillin. Plasmids were introduced into E. coli DH5o by using the RbCI method (Hanahan, 1983).
DNA was transformed into yeast cells by using the lithium acetate method (Ito et al., 1983).
Yeast genomic DNA was prepared as described (Wack et al., 1994). An overnight culture of yeast in 2.5 ml of YPD (-5 OD600 units) was collected and resuspended in TE buffer (10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0) with 0.5 M sorbitol. Cell wall was digested with -400 units of Lyticase (Sigma) and 5 gM dithiothreitol (DTT) at RT for 30 minutes. Spheroplasts were broken in 200 lI of 50 mM Tris-HCI (pH 8.0), 20 mM EDTA and 1.25% sodium dodecyl sulfate (SDS) at 650C for 20 minutes. After adding 100 pl of 3 M potassium acetate (pH 5.0), the mixture was centrifuged at top speed. DNA in the supernatant was precipitated with ethanol. RNA in the pellet was eliminated by enzymatic digestion with 2 gg/ml RNase A. DNA was again precipitated by ethanol.




Recovery of plasmid from yeast was done according to the method of Strathern and Higgins (Strathern and Higgins, 1991). Briefly, an overnight culture in 2 mls of YPD was collected and resuspended in 100 p.1 of TENS buffer (100 mM NaCI, 10 mM Tris-HCI, pH
8.0, 1 mM EDTA, and 0.1% SDS). The cells were broken by vortexing with 0.25 g of glass beads for one minute. The cell lysate was treated with phenol. The crude preparation containing plasmid DNA was obtained by treating the aqueous layer with a Geneclean kit (Bio 101). Using a gene pulser (BioRad), the recovered plasmid DNA (in 3 pl) was introduced into E. coli. DH5a.
Electro-competent E. Coli. DH5a cells were prepared as described below. An
overnight culture of E. coli. DH5a in 5 ml of 2X YT medium (1.6% Bacto-tryptone, 1% Bacto-yeast extracts and 0.5% NaCl) was diluted into 500 ml of 2X YT and grown at 370C for 2-3 hours. The cells were collected and washed twice with ice cold water. The cells were washed with ice cold 10% glycerol. Finally, the cells were resuspended in
1 ml of ice cold 10% glycerol. Aliquots of the cells were stored at -800C.
The restriction enzymes used in this study were purchased from New England Biolabs. Restriction digestions were performed according to the manufacturer's instructions.
DNA fragments were isolated as described below. After separation of DNA fragments on 0.8% agarose gels, a piece of Whatman GF/C filter paper on a piece of dialysis tubing membrane was inserted facing the band of interest. After electrophoresis for 20-30 minutes, the GF/C paper was removed, and the DNA on the paper was obtained by brief centrifugation. After adding an equal volume of phenol, the mixture was added onto a sephadex G-50 spin column. The DNA was recovered by spinning the column in IEC centrifuge at 1,000 rpm (~250g) for 10 minutes.
Southern blotting was done as described (Ausubel et al., 1993). Briefly, after
restriction digestion, the DNA samples were separated on a 0.8% agarose gel. The gel was treated sequentially with 0.25 M HCI for depurination, with denaturation buffer (1.5 M




NaC1, 0.5 M NaOH), and with neutralization buffer (3 M NaC1, 0.5 M Tris, pH7.0). Transfer of DNA from the gel to a piece of nitrocellulose paper was done with upward capillary transfer in 20X SSC buffer (3 M NaC1, 0.3 M Na3Citrate-2H20, pH 7.0). The nitrocellulose paper was then baked at 700C for 2 hours. Probes were labeled with [ax32P]dCTP by random oligonucleotide priming.
Manual DNA sequencing was done by using the dideoxy method (Ausubel et al., 1993). The double-strand template carried on a plasmid was denatured in 1 M NaOH at 370C for 5 minutes. The DNA with 2.0 pmol of a primer in 5X sequence reaction buffer (200 mM Tris-HCl, pH 7.5, 100 mM MgCI2, 250 mM NaC1) was incubated at 650C for 2 minutes, and slowly cooled to 300C. The annealed template-primer was mixed with 0.1 M DTT, 1.5 M dCTP, 1.5 M dGTP, 1.5 M dTTP, 5 gCi [a-35S]dATP, and 3 units of Sequenase 2.0 (Amersham). The mixture was incubated at 200C for 2-5 minutes. For each labeling reaction, 2.5 pl of ddATP, ddCTP, ddGTP, or ddTTP was added to four wells in a microwell culture dish, and incubated at 420C. The labeling mixture was added to each well and incubated at 420C for 2-5 minutes. Then 4 pl of stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol FF) was added to each well. After denaturation with steam for 2 minutes, the mixture in each well was separated on 8 M urea-6% polyacrylamide gel, and detected by autoradiography. The probes used for sequencing are listed as followings: P3, 4, 5, 6, 7, 8, 9, 10, 12, 21, 23, 24, 25, 26, 27, 28, 29 (see Table 4).
Construction of GAL-NOP2 Strain
Gene disruption was performed according to the method described by Rothstein (Rothstein, 1991). A diploid strain YPH501 was transformed with a NOP2::HIS3 disruption fragment (Figure 3). Correct integration of this fragment into one NOP2 locus in the His+ transformants (YSB 12) was confirmed by Southern blotting (not shown).




Figure 3. An intact copy of NOP2 gene is present in the cells from all of the viable spores. The restriction maps of NOP2, nop2::HIS3 fragment and the probe are as shown. The genomic DNA from the cells germinated from the two spores of one ascus (lane 1 and 2) and the four spores of another ascus (lane 3, 4, 5, and 6) was digested with HindIII and analyzed with Southern blotting.




Xhol EcoRI Hpal BamHI BgIll
2 1290 1966 2408 5856
rv, I I I I i NOP2 2681-4537 '
EcoRI Hndill Clal EcoRI Hindill EcoRI Sacl
-08 kb 954 1693 2630 4393 5012 6 kb
H BH H E
H Probe B

3.4 Kb-

2.2 Kb -

1 2 3 4

5 6




Figure 4. Nop2p is expressed in the cells from all of the viable spores. YBH9 and YSB 12 are the diploid strains with one NOP2 locus disrupted and replaced by LEU2 or HIS3, respectively. Total cellular proteins were extracted from the cells germinated from the four spores (a, b, c, and d) of the same ascus, separated on a SDSpolyacrylamide gel, and analyzed by Western blotting. The position of Nop2p is indicated. L: the wild-type haploid L4717. The band with a low molecular weight may be non-specific.




YBH9 (nop2::LEU2) YSB12 (nop2::HIS3)
a b c d a b c d L Nop2p f,.- PA o M4Poo ose




Figure 5. Disruption of NOP2 gene in a haploid L4717 strain. (A) The restriction maps of NOP2, nop2::LEU2 fragment and the probe. (B) The nop2::LEU2 strain, YBH3, was dependent upon the plasmid-carried NOP2 gene for viability. Serial dilutions of L4717, YBH12 and YBH3 were grown on SD medium with or without 5FOA. YBH12 was constructed by introducing pJPA40 (NOP2, URA3) into L4717. YBH3 was derived from the disruption of the NOP2 locus in YBH12 by nop2::LEU2 fragment. (C) Disruption of the NOP2 locus was confirmed by Southern analysis. Genomic DNA from L4717 (lane 1), and two NOP2 isolates (lane 2 and 3) and nop::LEU2 isolates (lane 4: YBH3, and lane 5: YBH4) carrying pJPA40 plasmid were digested with EcoRI and analyzed with Southern blotting. The 7.9-kb band corresponds to the linearized pJPA40 plasmid.




A
Xhol EcoRI Hpal BamHI Bgll
2 1290 1966 2408 5856
, NM 2681.,,M7
EcoRI Hindll Clal EcoRI Hindill EcoRI Sadcl
-08 kb 954 1693 2630 4393 5012 6 kb
C E H S
LEU2
H Probe B
I I
B C
+ 5-FOA 1 2 3 4 5
L4717-' 0 0 f *;
YBH12- 0 0 *
YBH30,- i 7.9
- 5-FOA
IID~ o -.1.3
L4717- O 1.3
YBH12. O
YBH3 .I




However, tetrad dissection revealed that all of the four spores from the same tetrad were viable with two His- and two His+ spores (not shown). To see if Nop2p was required for the viability of the cells from the spores, Western analysis with an affinity-purified polyclonal antibody APpAb3 to Nop2p was done (Figure 4, YSB 12). Nop2p was detected in the cells germinated not only from His- spores (lanes c and d) but also from His+ spores (lanes a and b) of the same tetrad (Figure 4). Genomic DNA was digested with HindII and analyzed with Southern blotting (Figure 3). The His- cells from the two spores exhibited a 3.4-kb band, but no 2.2-kb band (Figure 3, lanes 5 and 6), indicating the cells contained only one intact copy of NOP2. However, The His+ cells from another two spores showed not only 2.2-kb band but also 3.4-kb band (Figure 3, lane 3 and 4), suggesting that the cells contained copies of both the disrupted and the intact NOP2. Considering that this might be a strain-specific phenomenon, several other diploid strains (STX466, 9933D, YSB 11 and YJA501) were tested, but with similar results (not shown). Disruption with NOP2::LEU2 fragment also showed the same result (Figure 4, YBH9).
To avoid this sporulation-related problem, a disruption of the gene was made in a haploid strain in the presence of a plasmid-carried NOP2 gene. Part of the 5' non-coding region and 92% of NOP2 open reading frame (ORF) carried on pJPA40 plasmid was replaced by a BamHllHindII fragment containing the LEU2 gene, which was isolated from plasmid pJJ282. The NOP2::LEU2 disruption fragment (Figure SA) was then linearized with XhoI/SacI digestion. The wild type haploid strain L4717 cells were first transformed with NOP2 gene carried on the pJP40 plasmid (URA3 marker). The Ura+ transformant (YBH12) was viable in the presence of 5-FOA (Figure 5B), suggesting that the cells could survive by losing the URA3 plasmid. The YBH12 cells were then transformed with NOP2::LEU2 fragment (Figure 5A), which, if correctly integrated, should replace 92% of NOP2 ORF and part of the 5'-non-coding region with LEU2 gene. One of Leu+/Ura+ transformants (YBH3) was dead in the presence of 5-FOA, suggesting that NOP2 gene had been disrupted (Figure 5B). To further confirm the correct integration




of NOP2::LEU2 fragment at the NOP2 locus, genomic DNA from YBH3 and several other Leu+/Ura+ isolates was digested with EcoRI, and analyzed by Southern blotting (Figure 5C). In YBH3 (lane 4) and another isolate (lane 5), a 1.3-kb band was lost, but an extra 2.2-kb band was observed, indicating NOP2 gene was disrupted and replaced with LEU2 gene.
To generate a conditionally lethal nop2 allele, the NOP2 ORF was placed under the control of a repressible GAL10 promoter. First, a BamHI/XhoI fragment containing the NOP2 ORF was isolated from pJPA20 plasmid, and subcloned into plasmid pRD53. The SacI/XhoI fragment containing GAL-NOP2 was excised from this subcloned plasmid (pBH46), and cloned into pRS314 plasmid.
The GAL-NOP2 strain (YBH5) was constructed by using plasmid shuffling
technique (Sikorski and Boeke, 1991). The pBH47 plasmid was transformed into YBH3 strain. To replace the original plasmid (pJP40) with the second one (pBH47), the Trp+/Ura+ transformants were replica plated onto SGal plate containing 5-FOA. One of the colonies formed on the plate, YBH5, was used for the depletion analysis.
Cell Growth Curve and Viability Test
YBH5 cells were first grown at 300C in YPGal liquid medium until mid-log phase. The cells were then collected, washed and resuspended in water. Equal volume of the cells was transferred into YPD and fresh YPGal media, respectively. At different time points, cell densities were measured by using a spectrophotometer (Beckman) at A600. To keep the cells growing in mid-log phase, the cells were diluted into the fresh media whenever cell density reached 0.6-0.8 OD600 unit.
The six nop2 temperature-sensitive strains were grown at 250C in YPD, and then
shifted to 370C. Cell densities were measured as mentioned above. To test the cell viability after Nop2p depletion, the cells grown in YPD or YPGal medium were collected at the




different time points, diluted to 0.001 OD600 unit and then spread 20 pl (about 160 cells) onto YPGal plates. After incubated at 300C for 2 days, the number of the colonies formed on the plate was counted (Table 5).
Isolation of nop2 Conditionally Lethal Alleles
In vitro mutagenesis of the NOP2 gene. A low fidelity polymerase chain reaction (PCR) method (Muhlrad et al., 1992) was used to generate random mutations in NOP2 gene. A KpnI fragment containing NOP2 gene was cut out from pJPA29 plasmid and used as the template. Two oligonucleotides were used as the primers: Pl9A, complementary to the 5' end; and P16, complementary to the 3' end of the gene. Each of four PCR reactions was set up as followings: 20 ng DNA template, IX reaction buffer (50 mM Tris-HCI, pH 9.0, 50 mM NaCl, and 10 mM MgC12), 0.4 g.M of each primer, 0.1 mM each of three dNTPs with another dNTP at 0.025 mM, 1.25 units of Taq DNA polymerase (Promega), with or without 0.15 mM MnC12. Each reaction with a total volume of 25 p.l was overlaid with 100 pl. of mineral oil, and run in a Thermocycler (Coy) for 30 cycles (for each cycle: denaturation at 94oC for 90 seconds; annealing at 550C for 120 seconds; and extension at 720C for 180 seconds). The PCR products from the four reactions were then pooled and gel-purified. In this study, it was found that the majority of nop2 alleles generated in the presence of 0.15 mM MnCl2 were non-functional even at the permissive conditions, suggesting that Mn2+ caused a much higher error frequency in the PCR reaction.
Preparation of the gapped plasmid. pBH49 plasmid was cut with
AatllISnaBI, and the gapped plasmid was gel-isolated. The sequence homology between the gapped plasmid and the PCR products was -380 bp at the 5' end and -715 bp at the 3' end. It was found that with only 65 bp sequence homology, an efficient recombination still occurred (Muhlrad et al., 1992).




Construction of the stains containing only mutated nop2.
Approximately 140 ng of the PCR products and -60 ng of the gapped plasmid were cotransformed into YBH3 by the lithium acetate method. Muhlrad et al. found that homologous recombination between a gapped plasmid and a linear fragment following cotransformation into yeast is efficient (Muhlrad et al., 1992), so that in vitro ligation is not necessary. Plasmid shuffling was performed as described above. As controls, the gapped plasmid or the PCR products alone were also transformed into YBH3. Only 5-10 colonies/plate formed with the gapped plasmid alone, while no colony formed with the PCR products alone.
Isolation of conditionally lethal strains. Colonies formed in the presence of 5-FOA were replica plated onto SD medium, and incubated at 141C, 250C and 370C. Two other restrictive conditions, 6% ethanol or 3% formamide were also tested at 250C (Aguilera and Benitez, 1986; Aguilera, 1994).
Recovery of the plasmids. The plasmid DNA was recovered from the yeast cells, amplified in E. coli DH5a, and verified by restriction digestion. The ORF of nop2 alleles was cut out by digestion with EcoRI, gel-isolated, and subcloned into pBH49 plasmid at the same sites.
Mapping of the Mutations in the Six nop2 Alleles
The six nop2 alleles carried on pRS313 were manually sequenced and the predicted amino acid substitutions were located (see Figure 19). To identify the mutations contributing to temperature-sensitive (ts) phenotype, the multiple point mutations in each allele carried on pRS313 were separated into several groups by restriction digestion with BsgI, MfeI or SpeI. The restriction fragments containing the mutations were gel-isolated and exchanged with the fragments from NOP2. After ligation, the subclones were amplified in E. coli DH5ox. The subcloned plasmids containing one or several groups of




the point mutations were transformed into YBH3 strain. After plasmid shuffling, the phenotypic effect of the mutations contained in each subcloned plasmid was analyzed by incubating the cells under the restrictive condition (370C). The subcloned plasmids were constructed as described in Table 3.
SDS-PAGE and Immunoblotting
Total cellular proteins were extracted in the presence of trichloroacetic acid,
precipitated, and separated on 10.5% polyacrylamide gel by SDS gel electrophoresis (SDSPAGE), and analyzed by immunoblotting with affinity-purified polyclonal anti-Nop2p antibody (APpAb3) (de Beus et al., 1994). A monoclonal antibody (1G1) against the poly
(A) binding protein (PablP) (Anderson et al., 1993) was used to probe the same blot.
Measurement of Ribosomes and Polysomes
The levels of ribosomal subunits, monoribosomes and polyribosomes were measured as described previously (Baim et al., 1985). Cells in mid-log phase were collected, washed with lysis buffer (10 mM Tris-HC1, pH 7.4, 100 mM NaC1, 30 mM MgCl2) containing 100 jig/ml cycloheximide, and broken by vortexing with acid-washed glass beads. The lysate was centrifuged at 10,000g at 4oC for 5 min., and the supernatant was loaded onto 15%-50% sucrose gradient. The gradient was centrifuged at 40,000 rpm (285,000g) for 2.5 h at 40C in SW41Ti rotor (Beckman Instruments, Inc.). After centrifugation, the gradient was analyzed by using an absorbance detector (ISCO UA-5). The polysome curves were traced by using Freehand software.




Analysis of Pre-rRNA Processing by Pulse-Chase Labeling
Approximately 25 OD600 units of cells in the mid-log phase (OD600-0.5) were collected, and resuspended in 5 ml of the synthetic minimal media. The cells were pulse labeled with 250 gCi [methyl-3H]methionine or [3H]uracil at 250C for 3 min. A large excess of unlabeled methionine (5 mM) or uracil (2 mM) was added. At the different chase time points, I ml of cells was collected and centrifuged for 10 seconds. Cell pellets were immediately frozen in dry ice and stored at -700C for further analysis.
Total RNAs were extracted as described (Kohrer and Domdey, 1991). Briefly, cells were treated with 1% SDS and phenol at 650C for 5 min. After centrifugation, the supernatant was extracted with phenol::CHCl3 and CHCI3 sequentially. The RNAs were precipitated by using 0.3 M NaOAc plus 2.5 volume of 100% ethanol.
To increase the relative ratio of signal to noise, [3H]uracil labeled RNAs were passed through oligo (dT)-cellulose (Boehringer Mannheim). The supernatant was transferred to a fresh tube and the RNA was ethanol precipitated, washed and dried. The RNAs were separated on 1.2% agarose-glyoxal gel or 12% polyacrylamide-urea gel (Ausubel et al., 1993). The gel was treated with En3Hance (DuPont NEN), dried and exposed to X-ray film.
Northern Analysis of rRNAs
Total RNAs were extracted, separated on 1.2% agarose-glyoxal gel, and transferred to Hybon nylon membrane (Amersham). The blot was probed with the [32P] labeled oligonucleotides specific for the sequences in the internal transcribed spacer (ITS) 1 and 2, and in 18S and 25S rRNAs, and exposed to X-ray film. Before reprobing, the blot was submerged into boiling 0.5% SDS, cooled to room temperature, and washed with water. The oligonucleotides used as probes are listed in Table 4.




Primer Extension
Standard primer extension was performed as described below. Approximately 1 gg of RNA template and 1 pmol of primer in the buffer (25 mM Tris-HCI, pH 8.3, 25 mM KC1, 5 mM MgCl2, 5 mM DTT, 0.25 mM spermidine) were denatured at 700C for 10 min. and annealed at 420C for 20 minutes. Labeling was done for 1 minute at 420C in the presence of the followings: 0.5 mM each of dCTP, dGTP, and dTTP, 10 ACi of [a35S]dATP, 5 units of avian myeloblastosis virus reverse transcriptase (Promega) and 20 units of RNase Block II (Stratagene). Extension was done for 10 minutes at 420C after adding dATP to 1 mM. The reaction was stopped by placing the reaction mixture on ice and adding 10 mM EDTA, and the nucleic acids were precipitated with ethanol prior to electrophoresis in 8 M urea-6% polyacrylamide gel.
For concentration dependent pause (CDP) assay (Maden et al., 1995), the same procedure as the standard primer extension was performed, but with the concentration of dNTPs at 12.5 pM each, and 20 gCi of [a-35S]dATP during labeling. Extension was done with all four dNTPs at 4, 20, and 100 gM. For partial alkaline hydrolysis of RNA (Sirum-Connolly and Mason, 1993), 2 gg RNAs were digested in 20 gl of 50 mM Na2CO3, pH 9.0 at 900C for 6 minutes before standard primer extension.
In vitro Methylation Analysis
A 3.8-kb ApaI/BamHI fragment containing T7 or T3 promoter followed by 27S rDNA sequence was linearized from pBluescript KS+ or SK+. In vitro transcription was performed by using a commercially available kit (Promega). The reaction condition was set up according to the manufacturer's protocol. Both T3 and T7 RNA polymerase were successfully used. The reaction product was analyzed on a 0.8% agarose-glyoxal gel.




Nop2p was obtained by immunoprecipitation (IP) with a monoclonal anti-Nop2p antibody (D68) (Wu, P., B. Hong, J. S. Brockenbrough, and J. P. Aris, unpublished results). When the IP pellet was analyzed by SDS-PAGE and stained with coomassie blue, a band corresponding to the position of Nop2p was seen (not shown).
In vitro methylation reactions were performed as described (Segal and Eichler,
1991). The following components were contained in the reaction: 50 mM Tris-HCI (pH 8.0), 0.4 mM EDTA, 5 mM DTT, 0.4 mg/ml bovine serum albumin (BSA), 50 mM KCI,
2.0 p.M [3H]-S-adenosyl-methionine (NEN), -50 gM 27S rRNA, 40 units of RNase Block (Stratagene), and IP-purified Nop2p. The mixture with the total volume of 100 pl was incubated at 300C for one to two hours. Considering the possible effects of secondary structure, denaturation of 27S rRNA by boiling before adding to the reaction was also tested. The reaction product was treated with phenol/chloroform. In the presence of 20 ig of glycogen, the RNA was precipitated with 0.5 volume of 7.5 M ammonium acetate and
2.5 volume of 100% ethanol. The RNA was separated on a 0.8% agarose-glyoxal gel. The incorporated isotope was detected with fluorography.
RNase Protection Assay
RNase protection was done using a modification of a previously described method (Segal and Eichler, 1991). RNA was extracted from L4717 or YBH5 grown in SD for 8.5 hours, pulse labeled with [methyl-3H]methionine for 3 minutes, and chased for 4 minutes, as described above. [3H]labeled RNA from -7 OD600 units of culture (-30 pmol RNA) plus 300 pmol of oligonucleotide probe were denatured in 40% formamide, 40 mM PIPESHC1, pH 6.4,400 mM NaC1, 1 mM EDTA for 15 minutes at 850C. The oligonucleotides used for protection are as follows: oligo 6, oligo 7, and oligo 8 (Table 4). After cooling to 250C, single stranded RNA was digested with RNase A (20 gg/ml) and RNase T1 (3 gg/ml) freshly added to 300 mM NaCl, 10 mM Tris-HC1, pH 7.5, 5 mM EDTA.




Digestions were inactivated with SDS and proteinase K, supplemented with 10 gg yeast tRNA, extracted with phenol-chloroform, ethanol precipitated, washed with 70% ethanol, and dried. Samples were electrophoresed on 12% polyacrylamide/7M urea gels. Gels were fixed and treated with En3Hance (DuPont). Following fluorography, sections of dried gels were excised, digested with 1 ml of Scintigest (Fisher Scientific) at 370C overnight in tightly capped glass vials. Samples were neutralized by the addition of 100 pl of glacial acetic acid, mixed with Scintiverse E (Fisher Scientific), and subjected to scintillation counting for a sufficient length of time (0.5 8 hours per determination) to yield an error of 2% of the mean DPM value at a confidence level of 95%.
Immunoprecipitation Analysis
The buffers used in immunoprecipitation are listed as followings: IP buffer: 50 mM Tris-HCI (pH 7.5), 150 mM NaCI, 1 mM EDTA, and 2 mM NaN3. Lysis buffer-C: 50 mM Tris-HCl (pH 7.5), 150 mM NaC1, 1 mM EDTA, and 2 mM NaN3, 5 mM MgCl2,
0.1% NP-40 (Calbiochem), protease inhibitor cocktails in water (PIC-W) (1 mM benzamide, 0.5 gg/ml leupeptin, 1.5 pgM bestatin in ddH20), protease inhibitor cocktails in dimethylsulfoxide (PIC-D) (0.5 mM phenylmethylsulfonyl Fluoride, 1.5 gM pepstatin A,
1 gg/ml chymostatin in dimethylsulfoxide), 1 mM DTT, 5 mM vanadyl ribonucleoside complex. Pre-treatment buffer: 100 mM Tris-HCI (pH 9.0), 50 mM DTT, 5 mM EDTA. Lysis buffer-N: 20% Ficoll, 20 mM KPi, pH 6.5, 1 mM MgCl2, PIC-D and PIC-W. Cushion buffer: 30% Ficoll 400, 20 mM KPi, pH 6.5, 1 mM MgCl2, PIC-W, and PIC-D. Washing buffer: 50 mM Tris-HCI (pH 7.5), 500 mM NaCI, 1 mM EDTA, and 2 mM NaN3, 5 mM MgC12, 0.1% NP-40, PIC-W, and PIC-D.
Monoclonal anti-Noplp (A66) and anti-Nop2p (D68), and polyclonal anti-Nop2p (APpAb3) antibodies were used. Binding of antibodies to protein-G sepharose (Pharmacia) was performed as followings: about 20 pl protein-G sepharose beads were




washed with PBS buffer (2 mM KH2PO4, 8 mM Na2HPO4, 2.5 mM KC1, 140 mM NaC1, pH 7.2) plus 0.1% NP-40; after adding 5 pl. of either A66 or D68, or 8 gl of APpAb3, the mixture was incubated at the room temperature (-250C) for 60 minutes; the beads were then washed three times with PBS buffer plus 0.1% NP-40.
For each IP reaction, 5 OD600 units of L4717 cells in the mid-log phase were used. An overnight culture of AL4717 cells was diluted into fresh YPD to an OD600~0.1. After two doublings (OD600-0.4) at 300C, the cells were collected and washed with ddH20. For protein labeling, the cells were incubated in 10 ml of SD medium (without methionine) at 300C for 1 hour. The cells were then collected and labeled in 1 ml of SD medium with -25 gCi of [35S]methionine at RT for 30 minutes. For RNA labeling, the cells were incubated in 10 ml of low-phosphorus-YPD (LP-YPD) at 300C for 1 hour. Then the cells were labeled in 1 ml of LP-YPD with -30 pCi of 32P-phosphate at RT for 30 minutes.
For the whole cell lysate, labeled cells in 100 pl of lysis buffer-C were broken by vortexing with RNase-free glass beads for 5 minutes at 40C. The supernatant was used for IPs.
For nuclear preparation, labeled cells were treated with 100 pl1 of pre-treatment buffer. After washing with ddH20 and 1 M sorbitol, the cell wall was subjected to enzymatic digestion. For one OD60o of cells, 20 tg of Zymolyase 100T (ICN) and 1 g1 of glusulase (DuPont) were used. The digestion reaction was set up in 50 pl of 1 M sorbitol at RT for 30 minutes. The reaction was stopped by spinning for 30 seconds at RT at 7,000 rpm (4,000g), and the pellet was washed with cold I M sorbitol. Spheroplasts were homogenized in the lysis buffer-N using a Dounce homogenizer with 10 strokes at RT. The homogenate was centrifuged at 5,000 rpm (2,300g) in an SW50.1 rotor (Beckman) at 20C for 5 minutes. The supernatant was then loaded onto 1 ml of the cushion buffer. The nuclei in the supernatant were obtained by being centrifuged at 22,000 rpm (45,300g) in an SW50.1 rotor at 20C for 60 minutes. The nuclear pellet was resuspended in 0.5 ml of lysis




buffer-C and sonicated. After spinning at top speed in a microfuge at 4oC for 5 minutes, the supernatant was used for IP.
The supernatant from the whole cell lysate or from the nuclear preparation was mixed with the protein-G sepharose-antibody beads at 4oC for 1 hour with rotation. The beads were washed five times with washing buffer.
For protein analysis, the beads were resuspended in SDS-PAGE sample buffer (4% SDS, 20% glycerol, 0.1 M DTT, 83 mM Tris, 127 mM Tris-HC1, 0.001% bromophenol blue, pH 8.0), and boiled for 2 minutes. After a brief spin, the supernatant was loaded onto 10% SDS-PAGE, and analyzed by electrophoresis.
For analysis of RNA-protein interactions, the RNAs were extracted from the beads as follows. Beads were resuspended in 150 p l of IP buffer plus 1% SDS. In the presence of tRNA carrier, RNAs were extracted by 150 p.1 of phenol::CHCl3 at 370C for 15 minutes with vortexing for 1 minute every 5 minutes, and precipitated by ethanol. The extracted RNAs were separated on 7 M urea-6% polyacrylamide gel electrophoresis. The signals were detected by autoradiography.
Screening for High Copy Suppressors
A yeast library carried on 2 pm plasmid with URA3 marker was kindly given by Dr. Ruben Herriquez (Sandoz Pharmaceuticals AG). The library with an average of 10-kb inserts was prepared by Sau3A partial digestion and size fractionation. The library plasmids were transformed into E.coli DH5ac. Plasmid maxi-preparation was done as follows. An overnight culture of E.coli carrying plasmids was collected and treated with 1.25 mg/ml of Lysozyme (Sigma) in 40 mls of GTE buffer (25 mM Tris, 10 mM EDTA, 50 mM glucose, pH 8.0) at RT for 10 minutes. Cells were disrupted with 80 mls of 0.2 N NaOH plus 1% SDS on ice for 15 minutes. After adding 60 mls of 3 M KOAc (pH 5.0), the mixture was placed on ice for 15 minutes, and centrifuged at 4oC for 10 minutes at




5,500 rpm (4,000g) in Sorvall GSA rotor. DNA in the supernatant was precipitated by adding 0.6 volumes of isopropanol, and centrifuged at 4oC for 10 minutes at 4,000g. Pellet was dissolved in TE buffer, and mixed with 50 mls of 44% of CsCl plus 33% of ethidium bromide in quick-seal ployallomer tubes (Beckman). The mixture was centrifuged at 200C for 24 hours at 50,000 rpm (242,000g) in a VTi50 rotor. The bottom band containing plasmid DNA in CsC1 gradients was collected and treated with ddH20saturated 1-butanol. DNA was obtained by ethanol precipitation. The library was then transformed into each of the six ts strains. In this study, it was found that almost no colony was formed if the transformants were incubated at 370C immediately after transformation. Therefore, the transformants were incubated at 250C for 24 hours before shifting to 370C. Since yeast genome size is about 14 Mb, it can be estimated that with this library, a minimum of 1,400 colonies for each ts strain is required for covering the whole yeast genome. The library plasmids were transformed into each of the six ts strains. The plasmids were recovered from the colonies formed at 370C, and amplified in E. coli DH5ct. The suppression effect of the recovered plasmids was tested. Whether the recovered plasmids contained inserts was determined by restriction digestion with PstI, XbaI, NsiI and EcoRV, which cut the plasmid backbone. Presence of URA3 marker in the recovered plasmids was determined by digestion with EcoRV/NstI. Among about 100 plasmids recovered, only one contained a 3.7-kb insert. The SacI/BglII fragment containing 3.0-kb of the insert was then subcloned into pRS426 plasmid at the SacI/BamHI sites, and its phenotypic effect was re-tested. Sequencing of the insert was done with the reverse primer, which was complementary to the polylinker 5' upstream to the insert. Another primer, JA36, complementary to the sequence of 25S rDNA, was also used for sequencing. Sequencing with -20 and -40 primers complementary to the polylinker 3' downstream to the insert was not successful. Whether the suppression was allele-specific was analyzed by testing the phenotypic effect of the insert in each of the six ts strains.




Table 2. Yeast Strains
Strain Genotypic description Source

L4717

L4718
YBH3 YBH9 YBH12 YBH15 YBH16 YBHI7 YBH18 YBH20 YBH21 YBH23 YBH26 YBH27 YBH28 YBH29 YBH30 YBH31 YBH32 YBH33 YBH34 YBH35 YBH36

MATa, ade2, canl-100, his3-11,15,
leu2-3,-112, trpl-1, ura3-1, GAL+ MATa, ade2, canl-100, his3-11,-15,
leu2-3,-112, trpl-1, ura3-1, GAL+ L4717, nop2::LEU2, pJPA40 YPH501, nop2::LEU2 L4717, pJPA40 L4717, nop2::LEU2, pBH60 L4717, nop2::LEU2, pBH67 L4717, nop2::LEU2, pBH72 YBH 15, pJPA29 L4717, nop2::LEU2, pBH55 L4717, nop2::LEU2, pBH74 L4717, nop2::LEU2, pBH77 YBH20, pJPA29 YBH 16, pJPA29 YBH 17, pJPA29 YBH21, pJPA29 YBH23, pJPA29 L4717, nop2::LEU2, pBH49 L4717, nop2::LEU2, pBH56 L4717, nop2::LEU2, pBH57 L4717, nop2::LEU2, pBH58 L4717, nop2::LEU2, pBH59 L4717, nop2::LEU2, pBH61

C. A. Style G. R. Fink C. A. Style G. R. Fink This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study




Table 2. Continued
Strain Genotypic description Source

YBH37 YBH38 YBH39 YBH40 YBH41 YBH42 YBH43 YBH44 YBH45 YBH46 YBH47 YBH48 YBH49 YJA501 YPH501
YSB- 11 YSB 12 STX466
9933-13A 9933-20C
9933D

L4717, nop2::LEU2, pBH62 L4717, nop2::LEU2, pBH63 L4717, nop2::LEU2, pBH64 L4717, nop2::LEU2, pBH65 L4717, nop2::LEU2, pBH66 L4717, nop2::LEU2, pBH68 L4717, nop2::LEU2, pBH69 L4717, nop2::LEU2, pBH70 L4717, nop2::LEU2, pBH71 L4717, nop2::LEU2, pBH73 L4717, nop2::LEU2, pBH75 L4717, nop2::LEU2, pBH76 L4717, nop2::LEU2, pBH78 MATa/a, leu2A2, ura3-52, GAL2 MATal a, ade2-10 l1a, his3A200, leu2A1,
lys2-801a, trplA63, ura3-53, GAL+
Diploid from cross of L4717 and L4718 YSB- 11, nop2::HIS3 MATa/a, ade2-lo, his3A200, lys2-801a,
trplA, ura3-52, gal
MATa, his3A200, leu2-3,-112, trplA1,
ura3-52, GAL
MATa, his3A200, leu2-3,-112, trplAl,
ura3-52, GAL
Diploid from cross of 9933-20C and 9933-13A

This study

This study This study This study This study This study This study This study This study This study This study This study This study This study J. Anderson R. S. Sikorski
J. S. Brockenbrough J. S. Brockenbrough Yeast genetic stock center G. R. Fink G. R. Fink




Table 3. Plasmids
Plasmid Relevant functional DNA Comments

pAH6
pBH46 pBH47 pBH49 pBH55 pBH56 pBH57 pBH58 pBH59 pBH60 pBH61 pBH62 pBH63
pBH64

NOP2 HIS3 CEN6 GALIO-NOP2 URA3 CEN6 GALIO-NOP2 TRP1 CEN6 NOP2 HIS3 CEN6 nop2-3 HIS3 CEN6 nop2-3a HIS3 CEN6 nop2-3b HIS3 CEN6 nop2-3c HIS3 CEN6 nop2-3d HIS3 CEN6 nop2-4 HIS3 CEN6 nop2-4a HIS3 CEN6 nop2-4b HIS3 CEN6 nop2-4c HIS3 CEN6 nop2-4d HIS3 CEN6

Sadc fragment containing NOP2 cloned at the same sites of pRS313
BamHI/XhoI fragment containing NOP2 from pJPA20 cloned in the same sites of pRD53
SacI/XhoI fragment containing GALIO-NOP2 from pBH46 cloned in the same sites in pRS314
Derived from pAH6 with deletion of ClaI fragment from the upstream of NOP2
EcoRI fragment containing nop2-3 cloned at the same sites of pBH49
MfeI fragment from nop2-3 cloned at the same sites of pBH49
MfeI fragment from NOP2 cloned at the same sites of pBH55
SpeI fragment from pBH55 cloned at the same sites of pBH49
Spel fragment from pBH49 cloned at the same sites of pBH55
E.coRI fragment containing nop2-4 cloned at the same sites of pBH49
BsgI/ClaI fragment from nop2-4 cloned at the same sites of pBH49
BsgIl/ClaI fragment from NOP2 cloned at the same sites of pBH60
MfeI fragment from NOP2 cloned at the same sites of pBH62
MfeI fragment from nop2-4 cloned at the same sites of pBH49




Table 3. Continued
Plasmid Relevant functional DNA Comments

pBH65
pBH66
pBH67 pBH68 pBH69 pBH70 pBH71 pBH72 pBH73
pBH74 pBH75 pBH76

nop2-4g HIS3 CEN6
nop2-4h HIS3 CEN6
nop2-5 HIS3 CEN6 nop2-5a HIS3 CEN6 nop2-5b HIS3 CEN6 nop2-5c HIS3 CEN6 nop2-5d HIS3 CEN6 nop2-6 HIS3 CEN6 nop2-6a HIS3 CEN6
nop2-9 HIS3 CEN6 nop2-9a HIS3 CEN6 nop2-9b HIS3 CEN6

Derived from pBH62. MfeI fragment deleted, and the gapped plasmid ligated at the same site. ClallMfeI fragment from pBH49 cloned at the same sites. MfeI fragment from nop2-4 then cloned at the same site.
Derived from pBH49. MfeI fragment deleted, and the gapped plasmid ligated. Clal/MfeI fragment from pBH63 cloned at the same sites. MfeI fragment from nop2-4 then cloned at the same site.
E.coRI fragment containing nop2-5 cloned at the same sites of pBH49
MfeI fragment from nop2-5 cloned at the same sites of pBH49
MfeI fragment from NOP2 cloned at the same sites of pBH67
SpeI fragment from pBH67 cloned at the same sites of pBH49
SpeI fragment from pBH49 cloned at the same sites of pBH67
E.coRI fragment containing nop2-6 cloned at the same sites of pBH49
BsgI/MfeI fragment from nop2-6 (one of two MfeI sites lost from the mutation) cloned at the same sites of pBH49
E.coRI fragment containing nop2-9 cloned at the same sites of pBH49
MfeI fragment from nop2-9 cloned at the same sites of pBH49
MfeI fragment from NOP2 cloned at the same sites of pBH74




Table 3. Continued
Plasmid Relevant functional DNA Comments
pBH77 nop2-10 HIS3 CEN6 EcoRI fragment containing nop2-10
cloned at the same sites of pBH49
pBH78 nop2-10a HIS3 CEN6 BsgIl/MfeI fragment from nop2-10
(one of two MfeI sites lost from the mutation) cloned at the same sites of pBH49
pJJ282 LEU2 Jones, J. S. and L. Prakash
pJPA20 NOP2 EcoRI fragment containing NOP2
cloned into pBluescript SK+
pJPA29 NOP2 TRPI CEN6 Xhol/BglII containing NOP2 cloned
at Xhol/BamHI of pRS314
pJPA40 NOP2 URA3 CEN6 HpalIKpnI fragment containing
NOP2 plus 715 bp of 5' flanking sequence and 456 bp of 3' flanking sequence from pJPA20 cloned into pRS316
pRD53 GALl, 10 R. J. Deshaies
pRS426 21m URA3 T. Christianson




Table 4. Oligonucleotides

Name

Sequence

JA36 5'-GGTGGGTTAGTCGATCC-3'
Oligol (18S) 5'-AGCCATTCGCAGITCACTG-3' Oligo2 (ITS l-U) 5'-GCACAGAAATCTCTCACCGT-3' Oligo3 (ITS l -D) 5'-TCCAGTTACGAAAATTCTTG-3' Oligo4 (ITS2-D) 5'-GCCTAGACGCTCTCTTCTTA-3' Oligo5 (25S) 5'-TACTAAGGCAATCCGGTTGG-3' Oligo6 5'-CGCATAGACGT'AGACTCCTTGGTCCGTGTTTCAAGACGG-3' Oligo7 5'-CCCTATTAGTGGGTGAACAATCCAACGCTTACCG-3' Oligo8 5'-CGGTCTAAACCCAGCTCATGTTCCCTATTAGTGGG-3' Oligo9 (A) 5'-CCTTAGAGCCAATCCTTATC-3'
Oligo 10 (B) 5'-CTGTTGACGTGGAACCTTTC-3' Oligo 1l (C) 5'-CTGATGAGCGTGTATFCCGG-3' Oligol 12 (D) 5'-CAGGAACCAGCTACTAGATG-3' Oligo 13 (E) 5'-CACITCATTACGCGTATGG-3' Oligo 14 (JA22) 5'-CATCAGTAGGGTAAAACTAAC-3' Oligol5 (JA30) 5'-TGTTCTACTGGAGATTTCTG-3' Oligo 16 (JA31) 5'-TAGGGACAGTGGGAATCTCG-3' P3 5'-GCGGAGAACATCCAAC-3'
P4 5'-AGATCGGTCCATCTTCG-3'
P5 5'-GGTTGCATGTTCTCCGC-3'
P6 5'-CCTTGAAGACCAGAAGG-3'
P7 5'-CGAAGATGGACCGATCT-3'
P8 5'-GTACCGGAACATGGGGC-3'
P9 5'-TGAAGCAGGATTGGTGG-3'




Table 4. (continued)
Name Sequence
Pl0 5'-TCATCCACCAATCCTGC-3'
P12 5'-CTATGCTAACATGATGC-3'
P16 5'-TrCCTTCTACCGGTACC-3'
P19A 5'-GCCGGACGATAACGTGC-3'
P21 5'-CTAAGTCTCTCCTTCTG-3'
P23 5'-AAGAACAAGCAAGCCGC-3'
P24 5'-GTACCGAGAAGGACTTC-3'
P25 5'-GAATTCCCCTTGTGTTG-3'
P26 5'-AGCGAAAACACAACCTG-3'
P27 5'-GAATCAATGGCGGATAG-3'
P28 5'-TGCTCTAGATCCTCATG-3'
P29 5'-ACCGAATGTCAAGTTAG-3'
Reverse 5'-AACAGCTATGACCATG-3'
-20 5'-GTAAAACGACGGCCAGT-3'
-40 5'-GTTTTCCCAGTCACGGAC-3'




Time of Depletion
(Hour)

Table 5. Cell Viability after Nop2p Depletion
Colony Number
(Depleted) (Non-depleted)

Survivability(%

0 157 155 101
1 152 150 101
2 149 154 97
4 151 155 97
8 147 156 94
10 144 159 91
23 145 162 90
25 140 155 90




RESULTS

Depletion of Nop2p
Repression of NOP2 Impaired Cell Growth
When expression of a gene is repressed, the protein encoded by the gene will be depleted because of protein turnover and dilution by cell division. In this way, a deficit in the protein function will be revealed. To repress the activity of the GAL promoter and deplete Nop2p, YBH5 (GAL-NOP2) cells were shifted from YPGal to YPD medium. During the first 8 hours after the medium-shift, YBH5 cells grew at a similar rate in both media. However, after 8 hours' incubation, the cell growth in YPD medium was progressively impaired, as compared with that in YPGal medium (Figure 6A). The doubling time in YPD medium increased about fivefold from 2.5 hours at the medium-shift to 12.5 hours after the medium-shift for 28 hours. To confirm that Nop2p in the YBH5 cells grown in YPD medium was indeed depleted, Western blotting with an antibody to Nop2p was performed. At the 2 hour time point after medium-shift, the Nop2p level was significantly reduced. After 4 hours' depletion, Nop2p was not detectable (Figure 6B). The cytoplasmic poly(A) binding protein Pablp level was not affected during the incubation in YPD, suggesting that Nop2p was specifically depleted (Figure 6B). Since a significant depletion of Nop2p occurred before the cell growth slowed down, the cell growth was dependent upon the function of Nop2p.




Figure 6. The effect of Nop2p depletion on cell growth and the level of 25S rRNA. (A) Growth curves of YBH5 (GALNOP2) cells. The cells grown in YPGal were transferred to either fresh YPGal or YPD medium (0 time point). At the indicated time points, cell density was measured at A600. (B) Depletion of Nop2p was confirmed by Western analysis. YBH5 cells grown in YPD medium were collected at the indicated time points, and total cellular proteins were extracted and analyzed by Western blotting with an antibody to Nop2p. The same blot was re-probed with the antibody to the poly(A) binding protein Pablp. (C) Depletion of Nop2p caused a reduction in the 25S rRNA levels. Total RNAs were extracted from YBH5 cells grown in YPD medium for 0 and 28 hours, separated on an agarose-glyoxal gel, and stained with ethidium bromide.




0 lh 2h 4h 8h 1Oh 23h 25h Nop2p e-.,. -

Pablp-

- mm _

0 5 10 15 20 25 30
Time (hours)

0 28h

-25S 418S




Depletion of Nop2p Caused a Reduction in 25S rRNA and 60S Ribosomal Subunits
The nucleolar localization of Nop2p (de Beus et al., 1994) was a clue that it might be related to nucleolar function. The essentiality of this protein to cell growth and viability might be due to its involvement in the ribosome synthesis, one of the main functions in the nucleolus (Hadjiolov, 1985).
To investigate this possibility, the relative levels of 18S and 25S rRNAs in Nop2pdepleted cells were measured. Prior to Nop2p depletion (at 0 time point), the ratio of 25S and 18S rRNAs was close to 1:1 (Figure 6C). However, after 28 hours' depletion of Nop2p, 25S rRNA level was significantly reduced and the ratio of 25S to 18S rRNAs was much less than 1:1 (Figure 6C). Over 90% of YBH5 cells grown in YPD for 28 hours were still viable (Table 5). Therefore, the observed reduction of 25S rRNA was not caused by cell death. Since 25S rRNA is a structural component of 60S ribosomal subunit, these results suggest that Nop2p is involved in synthesis of the 60S ribosomal subunit.
To confirm the role of Nop2p in ribosome synthesis, the effect of Nop2p depletion on the levels of ribosomal subunits and polyribosome was analyzed. A typical, normal polysome profile was shown in the control L4717 cells (Figure 7A and B). In contrast, depletion of Nop2p for 4.5 hours caused a reduction in the level of 60S subunit relative to that of 40S subunit. The peak of 80S monomer, which consists of the 40S and 60S subunit associated with an mRNA, was accordingly lowered. Three small extra peaks appeared immediately following the peaks of 80S monomer and the first two polysome (Figure 7C). These extra peaks possibly represent halfmer polysomes, which contain the mRNA-associated polysomes plus a preinitiation complex consisting of 40S subunit with the initiation factors (Helser et al., 1981; Moritz et al., 1990). A similar abnormal polysome profile was observed when synthesis of 60S ribosomal subunit was aborted by depletion of large subunit proteins L1 or L16 (Moritz et al., 1990; Deshmukh et al., 1993).




Figure 7. Depletion of Nop2p causes a significant reduction in the level of the 60S ribosomal subunit YBH5 (C and D) and the wild-type L4717 (A and B) were shifted from YPGal to YPD medium, and collected after 4.5 hours (A and C) or 12.5 hours (B and D) after the medium shift. The ribosomal subunits, 80S monoribosome, and polyribosomes in the cell lysate were separated on sucrose density gradients and detected by measurement of A254 values. Halfmer ribosomes are labeled as H.




L4717 in YPD for 4.5 hours
80S

1.0
E : 0.5
C
0.5
0
C
1.0
E
c 0.5
In Nl

YBH5 in YPD for 4.5 hours
80S

Bottom

L4717 in YPD for 12.5 hours
1.0
80S
E 60S
to6
c
140.5
40S In 40B
N
Bottom

YBH5 in YPD for 12.5 hours

1.0
E
0.5
0n
0

40S

Bottom

Bottom




Since the growth rate of the Nop2p-depleted cells was similar to that of the non-depleted cells during the first 8 hours of growth in YPD, the aberrant polysome profile was not due to a secondary effect of slow growth. After the medium-shift to YPD for 12.5 hours, the level of the 60S subunit was significantly reduced while the 40S subunit accumulated (Figure 7D). The reduction of 80S monomer was more significant and the halfmer peaks were more obvious, suggesting that the shortage of the 60S subunit was much more severe after a longer period of depletion (Figure 7D). These observations indicate that Nop2p is required for synthesis of the 60S ribosomal subunit.
Nop2p is Required for Processing of 27SB Intermediate into 25S and 5.8S rRNAs
The fact that Nop2p depletion caused a reduction in 25S but not 18S rRNA
suggests that the protein might be involved in processing of pre-rRNA. To investigate this possibility, YBH5 (GAL-NOP2) cells grown in YPD for 8.5 hours were labeled with [methyl -3H] methionine, and chased with an excess of methionine. Since yeast rRNAs are methylated at a large number of sites soon after transcription (Brand et al., 1977), following the labeled methyl groups on the pre-rRNA to the mature rRNAs can reveal the intermediate steps of pre-rRNA processing. In the control L4717 cells, 35S pre-rRNA was almost immediately processed into 27S and 20S pre-rRNAs (Figure 8B). At 4 minutes of chase, most 27S and 20S pre-rRNAs were processed into 25S and 18S rRNAs, respectively. At 8 minutes of chase, almost all of these pre-rRNAs were converted to mature rRNAs (Figure 8B). However, in the Nop2p-depleted cells, 35S and 32S prerRNAs significantly accumulated at 2 minutes of chase. Even at 8 minutes of chase, a large amount of 27S pre-rRNA still accumulated with a significant reduction in the formation of 25S rRNA, which is consistent with the finding that the steady-state level of 25S rRNA was reduced after 28 hours of Nop2p depletion. Nevertheless, the processing of 20S to




Figure 8. Depletion of Nop2p results in a defect in pre-rRNA processing. YBH5(GAL-NOP2) (A) and L4717 (NOP2) (B) were shifted from SGal to SD for 8.5 hours, pulse-labeled with [3H-methyl]methionine, and chased with excess methionine for 0, 1, 2, 4 and 8 minutes. Total RNA was extracted, separated on an agarose-glyoxal gel, and detected by fluorography. Approximately one-third of the L4717 8-min sample was loaded on the gel.




A B
YBH5 (GAL-NOP2) L4717 (NOP2)
0 1 2 4 8min. 0 1 2 4 8min.
35S- -il
323
27S 1
25S S
208: EMIL
188so ..m a *




18S rRNA in Nop2p depleted cells was similar to that in the control cells (Figure 8A). These results suggest that Nop2p is involved in the processing pathway leading to 25S rRNA.
In this pulse-chase assay, the processing of pre-rRNA was evaluated by following incorporation of label into methyl groups, which had been transferred onto pre-rRNA. Therefore, the possibility had to be excluded that the aberrant processing might be actually a change in the methylation pattern. To do this, [3H]uracil was used to label the backbone of pre-rRNA for pulse-chase analysis. In the control L4717, 35S and 32S precursors were quickly processed (Figure 9B). Chasing of the 27S pre-rRNA to 25S rRNA in the control cells took a longer time than in [methyl -3H] labeling. This is possibly because the cell can re-use the labeled uracil resulting from the turnover of RNAs within the cell for a quite long time (Warner, 1991). In YBH5 depleted of Nop2p for 8.5 hours, a significant amount of 35S and 32S precursors were not processed even at 8 minutes of chase (Figure 9A). Compared with the control, a significant delay in the processing of 27S pre-rRNA to 25S rRNA was observed at 8, 16, and 32 minutes of chase, with the formation of 25S rRNA accordingly reduced (Figure 9A). Similar to what was observed in [methyl -3H] pulsechase labeling, the processing of 20S to 18S rRNA was not significantly affected by Nop2p depletion. These results confirm that Nop2p is required for processing of prerRNA.
To locate the specific processing step in which Nop2p might be involved, the
steady-state levels of pre-rRNAs and rRNAs were estimated by Northern analysis. With probes specific for regions in the ITS I and ITS2, and in the 18S and 25S rRNAs (Figure 10), the various processing intermediates can be recognized. Consistent with the results from the pulse-chase analysis, Nop2p depletion for 4.5 and 8 hours caused a significant accumulation of the steady-state levels of 35S and 32S precursors, as compared with the non-depletion cells (at 0 time point) and the controls YBH3 and L4717 (Figure 11 A, B, and C). With the probe (oligonucleotide 3) complementary to the 27SA but not 27SB




Figure 9. Pulse-chase labeling analysis of pre-rRNA processing by using [3H]uracil. YBH5 (GAL-NOP2) (A) and L4717 (NOP2) (B) were grown in SGal, shifted to SD for 8.5 hours, pulse-labeled with [3H]uracil, and chased with excess uracil for 0, 2, 8, 16, and 32 minutes. The samples were treated with oligo (dT)-cellulose before agarose-glyoxal gel electrophoresis.




YBH5 (GAL-NOP2)
0 2 8 16 32 ..-
m I

35S 32S-

27S ,25S-

20Sm,18S m,-

B
L4717 (NOP2)
0 2 8 16 32
e a




Figure 10. The yeast pre-rRNA processing pathway and the cleavage step requiring Nop2p function. Depletion of Nop2p caused the accumulation of 35S, 32S, 27SA and 27SB pre-rRNAs, but a significant reduction in the production of 25S rRNA. The results indicate that Nop2p functions at the processing step from 27SB to 25S rRNA (as indicated). The oligonucleotides used for Northern blotting (oligo 1, 2, 3, 4 and 5) and RNase protection (oligo 6, 7, and 8) are indicated as short bars below the 35S pre-rRNA.




5' ETS

AO Al D A2
35S
1 2

C2 C1

A3 B1S E
3

Processing AO Cleavage Al 32SI

Cleavage A2

20S 1
SCleavage D 18S E

27SA

Cleavage A3 Processing B2 Exonuclease A3 > B 1

Cleavage C2

Nop2p

7S
Exonuclease
C2 > E Cleavage C1

( S25S

7,8

F-

ITS 1

ITS 2

3' ETS

4 5 6

I

27SB

5.8S F




Figure 11. Northern analysis of steady-state levels of pre-rRNAs and mature rRNAs. Total RNAs were extracted from YBH5 (GAL-NOP2) collected at 0, 4.5, and 8 hour points after the medium-shift to YPD, and from YBH3 and L4717 grown in YPD medium. The RNAs were separated on an agarose-glyoxal gel, transferred to a nylon membrane, and probed with 32P-labeled oligonucleotides. (A) oligonucleotide 4 (ITS2 probe); (B) oligonucleotide 3 (ITS1 I probe); (C) oligonucleotide 2 (ITS I probe); (D) oligonucleotide 1 (18S probe) and 5 (25S probe) (see Figure 10).




Ppo
m o a ato a..m.,
(T) V ( T)V> TV T I TV

YBH3 L4717
o
YBH3 L4717

fl.o
PF

o
YBH3 L4717




pre-rRNA, the accumulation of 27SA pre-rRNA was observed in the Nop2p-depleted cells (Figure 1 IB). Nop2p depletion also caused a significant accumulation of 27SB pre-rRNA (Figure 1 A). The pathway leading 32S precursor to 23S and 27SB intermediates is a minor alternative one in wild type cells (Russell and Tollervey, 1992). Previous studies demonstrated that depletion of NopIp (Tollervey et al., 1991), or GarIp (Girard et al., 1992), or snoRNA U3 (Tollervey, 1987) caused the accumulation of 23S. Depletion of Nop2p had no significant effect on the level of this intermediate (Figure 11 C). The level of 20S pre-rRNA, a precursor for 18S rRNA, was similar among Nop2p-depleted cells and the control cells (Figure 1 IC). The sample loading was adjusted according to the levels of 25S and 18S rRNAs (Figure IID).
The above results indicate that Nop2p functions at the step of processing of 27SB to 25S rRNA (Figure 10). If this is true, then the formation of 5.8S rRNA should also be affected by Nop2p depletion, since 27SB is the common precursor for 25S and 5.8S rRNAs (Figure 10). To test this assumption, the processing of 5.8S rRNA was evaluated by pulse-chase analysis. At 10 and 30 minutes of chase, the formation of 5.8S rRNA was reduced in YBH5 cells depleted of Nop2p for 8.5 hours, as compared with that in the control LA717 (Figure 12A). Approximately equivalent loading was estimated by staining the gel with ethidium bromide (Figure 12B). Since 5S rRNA is transcribed independently by RNA polymerase II, its level should not be affected by the defect in the processing of the large rDNA transcript (Raue and Planta, 1991). A similar rate of the formation of 5S rRNA was observed in YBH5 and the control L4717 cells, suggesting that Nop2p depletion specifically affected the formation of 5.8S rRNA (Figure 12A).
In summary, the results from the pulse-chase analysis and Northern blotting indicate that Nop2p is required for processing of pre-rRNA, specifically at the step of conversion of 27SB pre-rRNA to 25S and 5.8S rRNAs. However, the formation of 18S rRNA from 20S precursor is relatively independent of Nop2p function.




Figure 12. Depletion of Nop2p causes a defect in the synthesis of 5.8S rRNA. (A) YBH5 (GAL-NOP2) and L4717 (NOP2) were grown in SGal, shifted to SD for 8.5 hours, pulse-labeled with [3H]uracil, chased with excess uracil for 0, 3, 10 and 30 minutes. Total RNAs were extracted, separated on a 12% polyacrylamide-urea gel and detected by fluorography. (B) To estimate the loading, the same gel was stained with ethidium bromide. The arrow indicates 0.15-kb RNA standard.




YSH5 (GAL-NOP2) 0 3 10 30

L4717 (NOP2) 0 3 10 30




Mutational Analysis of Nop2p
Another approach to investigate the function of a protein is to make mutations in the protein. A protein containing point mutations may assume a non-functional conformation at a restrictive condition. If the gene is essential, the mutations will cause a lethal phenotype at a restrictive condition, but not at the permissive condition. At a certain restrictive condition, global changes in cellular processes (cytoskeleton, protein synthesis, replication, transport, etc.) may occur, also causing cell death. However, this possibility can be excluded if the lethal phenotype can be rescued by the gene encoding this protein. One advantage of this approach is that all of the mutated protein generated at a restrictive condition will be non-functional, while it takes a much longer period of time to deplete a protein. Therefore, a deficit in the function will be caused more likely by the mutated protein than by secondary effects. Another advantage is that different alleles of a gene may cause different phenotypes, which helps to reveal multiple functions of the gene product (Huffaker et al., 1987).
Isolation of the Six Temperature-Sensitive nop2 Alleles
In this study, random point mutations in NOP2 were generated in vitro by using a low fidelity PCR method (Muhlrad et al., 1992). The phenotypes of the cells containing only the mutated nop2 alleles were analyzed under three restrictive conditions. One is temperature, which has been widely used to isolate conditional mutants (Huffaker et al., 1987; Tollervey et al., 1993). At low (14'C) or high (37C) temperatures, it may be more demanding for a protein to maintain its functional conformation. Mutation at critical site(s) of the protein can disrupt the functional conformation, causing a mutant phenotype at either high (Ts) or low (Cs) temperatures. Recently, formamide and ethanol were found to be very useful for isolation of conditional mutants (Aguilera and Benitez, 1986; Aguilera,




1994). The chemical groups (C=O, OH, NH) present in formamide and ethanol may compete for noncovalent bonds with the amino acid residues of the protein. Mutation in a protein may weaken the non-covalent bonds formed among the amino acid residues because the amino acid substitution may alter charges or hydrophobicity at the local region. In the presence of formamide or ethanol, the functional conformation of a mutant protein may be disrupted, resulting in either formamide-sensitive (Fs) or ethanol-sensitive (Es) phenotype.
Two mutagenic PCR conditions were tested to generate mutations in the NOP2. One includes the addition of 0.15 mM of MnC12 and a bias in the nucleotide pools. Another includes the bias in the nucleotide pools only. The PCR products and the gapped plasmid pBH49 were cotransformed into YBH3 cells. After plasmid shuffling (see MATERIALS AND METHODS), the effects of the mutations were tested. With the first condition, only very few colonies (an average of 40-50/plate) formed at 25C (not shown), suggesting that most of the nop2 alleles were non-functional even under the permissive condition. An estimate of 10,000 colonies carrying the PCR products from the first condition was screened for Ts and Cs phenotypes. However, no conditionally lethal strain was found. With the second condition, an average of 150-200 colonies/plate formed at 25C. In order to increase the chance of isolating conditionally lethal strains, two additional restrictive conditions, 3% formamide and 6% ethanol, were used. A total of 22,000 colonies carrying the PCR products from the second condition was screened. Some Ts, Cs, and Fs/Ts candidates were isolated, but no Es or Fs only candidate was found (Table 6). To exclude the possibility that the phenotypes might be caused by mutation in other genes on the chromosomes, the plasmids in these candidates were recovered, and their phenotypic effects were tested again in the same genetic background (Table 6). Among the twenty-six Ts candidates, the majority (73%) were caused by the mutation on the chromosomes. No Cs phenotype was found to be plasmid-borne. The plasmid recovered from the Fs/Ts candidate still exhibited the phenotypes (Table 6). To make sure that it was




Table 6. Screening for Conditionally Lethal nop2 Alleles
Phenotype Candidates Plasmid-borne phenotype ORF-bomrne phenotype
Ts 26 7 5
Cs 2 0 0
Fs/Ts 1 1 1
Es 0 0 0

Ts: temperature-sensitive. Cs: cold-sensitive. Fs: formamide-sensitive. Es: ethanol-sensitive. ORF: open reading frame.




the mutation in the protein that caused the phenotypes, the open reading frame (ORF) of the alleles was subcloned and its phenotypic effect was tested again. A total of six conditionally lethal alleles were isolated. All six alleles conferred the cells with the temperature sensitive phenotype, while they still supported cell growth to some degrees at the permissive temperature (25*C) or at 30'C (Figure 13). In liquid medium, 8 hours after the temperature shift, the growth curve of the six Ts strains already reached a plateau, while the wild type L4717 still grew in log-phase (Figure 14). Interestingly, nop2-10 strain expressed not only Ts, but also Fs at 25C (Figure 15, nop2-10), which suggests that the mutation at an additional site(s) of this allele caused this additional phenotype that was not expressed in the other Ts strains. The conditionally lethal phenotype of the six Ts strains was rescued by another plasmid carrying the wild type NOP2 gene (Figure 15 and not shown). This confirms that the phenotype was caused by mutation in NOP2.
To exclude the possibility that the phenotype might be due to rapid protein
degradation caused by the mutations, immunoblotting analysis of the mutated versions of the protein was performed. After 6 hours at 37C all of the six mutated versions of the protein were present, though the amounts were relatively lower than the two controls (LA717 and YBH3) (Figure 16). This result suggests that the mutant proteins were relatively stable. Compared with the two controls (L4717 and YBH3), there was no change in protein mobility of nop2-3 and nop2-4. However, nop2-5 protein moved faster than the other mutants and the controls (Figure 16). Sequencing of this allele revealed that there was an amber mutation near the 3' end of the ORF, causing a truncation of 14 amino acids at the C terminus (see Figure 19). There was also a change in the protein mobility of nop2-6, nop2-9 and nop2-10 (Figure 16), which may be due to changes in charge or modification of the mutant proteins.
The above results indicate that the phenotypes of the six nop2 alleles isolated are due to the mutations in the ORF of the gene and that the amino acid substitutions in the mutant proteins result in disruption of the functional conformation of the protein at the




Figure 13. The effect of temperature on the growth of the six nop2 temperaturesensitive (ts) strains. Serial five-fold dilutions of the cells of L4717, YBH3, and the six nop2 ts strains (nop2-3, nop2-4, nop2-5, nop2-6, nop2-9 and nop2-10) were grown on YPD plates at 250C, 300C or 370C.




250C

L4717 O YBH3 O nop2-3 O nop2-4 nop2-5 **
nop2-6 0O nop2-9 nop2-10 .

300C
**
@0OOA
-@.*O'

370C
OOO4 *




Figure 14. Growth curves of the six nop2 ts strains. The cells of L4717 and the six nop2 strains (nop2-3, nop2-4, nop2-5, nop2-6, nop2-9 and nop2-10) were grown in YPD at 250C and shifted to 370C (at 0 time). At the indicated times, the cell densities were measured (A600).




76
1000
-*- nop2-3
........ ........ nop2-4
100 ----0---- nop2-5
" ~-------- nop2-6
- -EB- - nop2-9
10- --- -- nop2-10
.....**L4717
---------------------------------------A------------- --------------------"" ' ...d .. ........... ..... ./
. 1 .- .....
I. B

0 5 10 15 20 25
Hours at 370C




Figure 15. Mutations in NOP2 caused temperature sensitive and formamide sensitive phenotypes. The cells of nop2-9 and nop2-10, and the corresponding "rescue" strains (carrying pJPA29 plasmid) were streaked on YPD plates with or without 3% formamide, and incubated either at the restrictive (370C) or the permissive (250C) temperature as indicated. The growth of wild type L4717, and YBH3 (carrying plasmidborne NOP2) are shown as controls.




nop2-9 iop2
Alele /
.: YBH3 rescue 370C
nop2-10
250C 25oC
- FA +FA

250C

370C
- FA




Figure 16. Mutant nop2 proteins are relatively stable. The cells of L4717, YBH3 and the six nop2 ts strains (nop2-3, nop2-4, nop2-5, nop2-6, nop2-9 and nop2-10) were grown in liquid YPD at 370C for 6 hours. Total cellular proteins were extracted and analyzed by Western blotting with a polyclonal antibody to Nop2p. The position of Nop2p is indicated. Non-specific bands are labeled with stars.




80
rl
cf)
m nop2 alleles
3 4 5 6 9 10
Nop2pp-




restrictive conditions. Since nop2-10 allele caused Fs, in addition to Ts phenotype, several structural domains may exist in the protein. Mutation in some domains may result in Ts phenotype only, as was shown in the other five nop2 alleles, while mutation in other domains may cause Fs or both Ts and Fs phenotypes. The Ts or Ts/Fs phenotypes are most possibly caused by the direct effects of mutation in NOP2 gene, though it is also possible that mutation in NOP2 may make the cells extremely "sick" and that a second "hit" with heat or formamide may actually cause cell death.
Characterization of the Six nop2 Alleles
One advantage of using a mutational analysis approach is that it can reveal multiple functional domains in a protein. Since each of the six nop2 alleles contains multiple point mutations, analysis of the effects of these different mutations would help understand more about the protein's function.
Each allele was analyzed for its effect on the synthesis of ribosomes. The six Ts
strains and the control YBH3 strain (carrying plasmid-borne NOP2) were incubated at 370C for two hours, and the levels of ribosomal subunits and polysomes were measured. In the control YBH3, incubation at 370C did not cause any significant change in the polysome profile (not shown). In contrast, even at the permissive temperature (25C), all of the Ts strains, except nop2-3, already exhibited a significant change in the polysome profile, including a reduction in the peaks of the 60S subunit and the 80S monosome, a relative accumulation of 40S subunit, and appearance of halfmer polysomes (Figure 17). At 370C for 2 hours, a more severely abnormal polysome profile was seen in all of the six Ts strains (Figure 17). However, the degree of the severity in the defect appeared to vary among the six Ts strains, with nop2-10 strain affected most. When YBH3 was incubated in the medium containing 3% formamide at 25'C for two hours, the peaks of both 40S and 60S subunits were proportionally reduced. However, there was no change in the peaks of 80S




Figure 17. Mutation in NOP2 impairs the synthesis of 60S ribosomal subunit. The six nop2 ts strains (nop2-3, nop2-4, nop2-5, nop2-6, nop2-9 and nop2-10) were grown in YPD at the restrictive (370C) or permissive (250C) temperature for 2 hours. nop2-10 and YBH3 were grown in YPD containing 3% formamide at 250C for 2 hours. YBH3 was grown at 250C. Cell lysates were analyzed on sucrose density gradients. The ribosome subunits, monoribosome and polyribosomes were measured as A254 values.




nop2-4 250C

Bottom nop2-3 370C

Bottom Bottom
nop2-10 250C nop2-10 370C

Bottom

nop2-5 370C

Bottom nop2-10 3% FA

Bottom

nop2-6 370C

Bottom YBH3 3% FA

nop2-9 370C

Bottom YBH3 250C

Bottom

nop2-3 250C

nop2-5 250C

nop2-6 25C

nop2-9 25C

Bottom

Bottom

Bottom

Bottom




monomer and polysomes, and no halfmer polysome was seen (Figure 17). When incubated in the medium containing 3% FA at 25'C, only nop2-10 strain showed a similar abnormality that was seen when incubated at 37C (Figure 17 and not shown). The results confirm that Nop2p is required for the synthesis of 60S ribosomal subunit. Since there are different degrees of severity in the defect caused by these six alleles, and only nop2-10 strain showed the formamide-sensitive phenotype, it is possible that several regions within Nop2p may be required for the functional conformation.
The effects of the six Ts alleles on pre-rRNA processing were evaluated by pulsechase labeling analysis. After 2 hours at 37C, all of the six Ts strains (nop2-10 not shown) exhibited a significant accumulation of 35S and 32S precursors. At the same time, the formation of 25S rRNA was significantly delayed and reduced (Figure 18). In the control YBH31 (carrying plasmid-bome NOP2) grown under the same conditions, the processing of pre-rRNA was rapidly completed (Figure 18). There was no accumulation of 20S pre-rRNA and the formation of 18S rRNA was not significantly affected in the six Ts strains at 37C (Figure 18). When grown in 3% formamide medium at 25C, nop2-10 strain also exhibited a similar defect in the processing that was observed at 37C (Figure 18). However, when grown in the medium without 3% formamide at 250C, the processing in nop2-10 appeared to be similar to that in the control YBH31 (Figure 18). These results are consistent with those from Nop2p depletion analysis. However, the 27S processing intermediate rapidly disappeared in nop2-4 and nop2-5 strains, though the formation of 25S rRNA in these two strains was also reduced (Figure 18). In all of the other Ts strains, accumulation of this intermediate was significant, even at 8 minutes of chase (Figure 18). Since Nop2p was involved in the methylation of only one site on 25S rRNA sequence (see below), It is most possible that the 27S intermediate in nop2-4 and nop2-5 strains was rapidly degraded. These results confirm that Nop2p is required for the processing pathway leading to 25S rRNA. The fact that 27S processing intermediate did not accumulate in nop2-4 and nop2-5 strains suggests that the region(s) mutated in nop2-4 and nop2-5 alleles




Figure 18. Mutation in NOP2 causes a defect in pre-rRNA processing. YBH31 (carrying plasmid-borne NOP2), nop2-3, nop2-4, nop2-5, nop2-6 and nop2-9 were grown in SD medium, and collected after temperature shift to 370C for two hours. nop2-10 was grown in SD medium with or without 3% formamide at 250C for two hours. The cells were pulse-labeled with [methyl-3H] methionine, and chased with excess methionine for 0, 2, 4, 8 and 12 minutes. Total RNA was separated on agarose-glyoxal gel, and detected by fluorography. The star indicates the position of 23S pre-rRNA.




YBH31
0 2 4 8 12

35S 32SM-

nop2-3
0 2 4 8 12

nop2-4
0 2 4 8 12

nop2-5
0 2 4 8 12

...-

- m Nme 1
eom

g l U-. an mm

d m am

nop2-6
0 2 4 8 12

nop2-9
0 2 4 8 12

nop2-10 + FA (250C) 0 2 4 8 12

nop2-10 (25oC)
0 2 4 8 12

35Sm'328 *

27S._- '
2S- # 18S- s

Sdam

mm
a.

amb

27SP. ~ 25S -

208S- am 18S-

# te




was required for the stability of 27S intermediate. It is possible that certain region(s) in Nop2p may be involved in the association of other proteins or snoRNPs with 27S intermediate and that disruption of such association may subject 27S intermediate to a rapid degradation.
Structure and Function Analysis of Nop2p
Analysis of the sites of point mutations in a protein can reveal the relationship among the protein primary sequence, conformation and function. To determine the changes in the primary structure of the mutant Nop2p, all of the six alleles were sequenced, and mutation sites were located (Table 7). Most of the point mutations caused amino acid substitutions (missense mutations) (Figure 19), while some mutations did not change amino acid residues (silent mutations) (Table 7). Only one point mutation resulted in a non-sense mutation (K605 to amber mutation in nop2-5) (Figure 19). Muhlrad et al. reported that the most common base changes are A/T>G/C transitions (Muhlrad et al., 1992). In this study, A/T>G/C and G/C>A/T transitions comprised 57% of the total base changes in the six nop2 alleles, with G/C>A/T the most common change (33%) (Table 8). Each of the six alleles contained from 7 to 12 missense mutations. In nop2-10, all of seven point mutations were located in the C-terminal half of the protein sequence (from aa 254 to aa 560). In contrast, the mutations in all the other five alleles spanned the whole protein sequence. According to the sequencing data, replacement of some amino acid residues caused changes in charge or polarity at these sites. For example, 1309K change in nop2-3 made this site become positively charged (Figure 19). Protein secondary and tertiary structures depend on the non-covalent interactions among the side chains of amino acid residues. Therefore, it is likely that the amino acid substitutions in the mutant Nop2p make the functional conformation of the protein unstable or disrupted at the restrictive conditions.




Table 7. PCR-Generated Point Mutations in NOP2 Alleles Mutationst
nop2-3 A145>G, T197>A*, G531>A, A778>G, T902>A*, T925>A,
T940>C*, A1103>T, C1202>T*, G1291>A, G1408>A, A1640>C nop2-4 G207>A, A252>G, A256>T, A286>T, G427>T, G520>A,
T729>C, A829>G, C839>T*, G915>A*, T1045>C, G1131>A,
C1489>T
nop2-5 G192>A, G561>A, A826>G, C1062>A, T1193>C*, G1131>A,
A1396>T, G1647>A, A1749>C, G1758>T, A1812>T#
nop2-6 A42>T, A322>T, G437>A, G465>A, T594>A*, A617>G*,
T720>A, T792>A, CA944>TT, Cl005>T*, T1319>A, A1450>T,
C1547>T*, G1718>T*
nop2-9 A86>G*, AA247>TT, C350>T*, T368>G, A450>G, T473>A*,
G504>A, T520>C, C 162>A, G 174>A, C1347>T, A1529>T nop2-10 T692>C*, A725>T*, T759>A, G898>A, C943>A, G972>T,
A1 107>T, T1292>C*, G1494>A, C1675>A
t Numbers indicate positions of missense mutations in NOP2 sequence.
* Silent mutations
# Nonsense mutation




Table 8. Types of Base Changes in the Six nop2 Alleles Type of base change % observed
Transitions
A/T>GIC 24%
G/C>A/T 33%
Transversions
A/P>T/A 29.3%
A/Tr>C/G 0.26%
G/C>T/A 11.6%
G/C>C/G 0




Figure 19. Amino acid substitutions in nop2 mutants. Nop2p with a total of 618 amino acid residues is shown as a box from residue 1 (N-terminus) to residue 618 (C-terminus). The amino acid changes (indicated with arrows) and the corresponding positions in the Nop2p are shown for the six nop2 alleles (nop2-3, nop2-4, nop2-5, nop2-6, nop2-9, nop2-10 ). Amb: amber mutation. Motif I (SAM binding domain): from residue 347 to 363; motif II: from residue 415 to 430; motif HIII: from residue 468 to 484.




Summary of Amino Acid Changes in nop2 Mutants

II
H

III
r0

N C
1 100 200 300 400 500 600 618

I V K K309 A314 N368

G
D431

G
D470

0 I H547 T549

D R K E A M
N70 G85 186 V108 S143 1174

E
K65

E
K188

F K G I A
L244 R279 S306 T349 T378

N
S276

A A D355 T378

P
L497

N
1466

G T 0 K605 S550 A584 Y587 Amb

M E S
1146 K156 T199

Y F N241 1263

K N K V M
L83 K123 E151 M169 T174

P
L315

A R D388 H392

S G P G T
T254 S300 0315 C325 S370

E
V484

P V K
L449 M482 N510

V
1499

nop2-3

K
R49

A
T178

E
G260

nop2-4

nop2-5

nop2-6
T
S15
nop2-9

E
G 108

nop2-10 (ts and fs)

L
F573

A
E560




Since the random mutagenesis PCR method was used in this study, it was
predictable that multiple mutations spanning the whole protein sequence would occur. This was confirmed by the results from sequencing of the six nop2 alleles (Table 7). In many cases, Ts phenotype is caused by one or a few point mutations at critical sites ("hot spots"). To determine the mutations responsible for the phenotype, several restriction sites within the ORF of each nop2 allele was used to separate the mutations in each allele into several groups. The phenotypic effects of each group or the combination of several groups of mutations were analyzed.
nop2-3 allele. In this allele, the two point mutations within MfeI fragment are not located in any of the motifs (Figure 19), and did not cause the phenotype (Figure 20, nop2-3a). Another two point mutations located 3' to SpeI site were not required for the phenotype (Figure 20, nop2-3c).
nop2-4 allele. In this allele, one BsgI and two MfeI sites were used to separate the allele into four parts. The effect of the mutations in each part was tested. The six point mutations upstream to BsgI site did not cause any detectable phenotype at 37C (Figure 20, nop2-4a). However, the six point mutations downstream to BsgI site caused the Ts phenotype (Figure 20, nop2-4b). These six point mutations were then separated into three groups. Each group independently or the combination of the two groups did not cause the Ts phenotype (Figure 20, nop2-4c, nop2-4d, nop2-4g and nop2-4h). However, the effect of each group seemed to be additive (Figure 20, nop2-4g). The mutation in the SAM binding domain (1304T) (Figure 19) with another mutation in the MfeI fragment did not cause the Ts phenotype (Figure 20, nop2-4d). The results suggest that at least the three point mutations located in the three regions of Nop2p are required to cause the phenotype. Mutation in one of them may not significantly change the conformation.
nop2-5 allele. The two point mutations in MfeI fragment of this allele caused the phenotype, though this allele was a little leaky (Figure 20, nop2-5a). Since A378T also occurs in nop2-4, but did not cause Ts phenotype, it may be A355 to D change that caused




Figure 20. Mapping of the mutations in the six nop2 alleles. The point mutations (labeled as asterisk in boxes) in each of the six nop2 alleles were separated into groups using the restriction sites BsgI, MfeI, or Spel (see METHODS AND MATERIALS for detailed descriptions). The nop2 alleles thus constructed are shown as the boxes (the left panel). Serial dilutions of the cells carrying the corresponding nop2 alleles were grown on the SD plates at 25'C (the middle panel) or 37'C (the right panel). YBH31 strain carrying the plasmid-borne NOP2 (labeled as NOP2) is shown as a control.




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CHARACTERIZATION OF A YEAST NUCLEOLAR PROTEIN Nop2p BY BO HONG 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 UNIVERSrTY OF FLORIDA 1997

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This work is dedicated to my parents

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ACKNOWLEDGMENTS I would especially like to thank my mentor, Dr. John P. Aris, for his guidance and training. I would like to extend my gratitude to my committee members, Dr. William A. Dunn, Dr. Alfred S. Lewin, and Dr. Maurice S. Swanson, for their encouragement and support. I would also like to thank the faculty and staff at the Department of Anatomy and Cell Biology for providing a stimulating intellectual environment for my graduate training. Dr. J. Scott Brockenbrough deserves my special thanks for his assistance and friendship. Most important, I would like to thank my wife Liu for her understanding and support. in

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TABLE OF CONTENTS gage ACKNOWLEDGMENT hi ABSTRACT vi INTRODUCTION 1 The Nucleolus and Ribosome Synthesis 1 Processing of Pre-rRNA 3 Modification of rRNAs and Ribosomal Proteins 6 Functions of the Nucleolar Proteins 9 Functions of SnoRNAs 17 Interaction of Nucleolar Proteins and SnoRNAs 18 MATERIALS AND METHODS 20 Strains, Media, and Microbiological Methods 20 DNA Methods 21 Construction of GAL-NOP2 Strain 23 Cell Growth Curve and Viability Test 31 Isolation of nop2 Conditionally Lethal Alleles 32 Mapping of the Mutations in the Six nop2 Alleles 33 SDS-PAGE and Immunoblotting 34 Measurement of Ribosomes and Polysomes 34 Analysis of Pre-rRNA Processing by Pulse-Chase Labeling 35 Northern Analysis of rRNAs 35 Primer Extension 36 In vitro Methylation Analysis 36 RNase Protection Assay 37 Immunoprecipitation Analysis 38 Screening for High Copy Suppressors 40 RESULTS 50 Depletion of Nop2p 50 Mutational Analysis of Nop2p 69 Nop2p and Methylation 96 Interaction of Nop2p with other Macromolecules 109 IV

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DISCUSSION 117 Nop2p is Essential for Cell Viability and Growth 117 Nop2p is Required for Ribosome Synthesis 118 Interaction of Nop2p with Other Macromolecules 122 The Mechanism by Which Nop2p Functions 123 REFERENCES 129 BIOGRAPHICAL SKETCH 137

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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 CHARACTERIZATION OF A YEAST NUCLEOLAR PROTEIN Nop2p By Bo Hong August 1997 Chairman: John P. Aris, Ph.D. Major Department: Anatomy and Cell Biology The main function of the nucleolus is the synthesis of ribosomes, which involves a series of complicated events, including processing and modification of pre-rRNA. It has been proposed that nucleolar proteins play important roles in these events. However, only a few nucleolar proteins have been identified so far, and the detailed molecular mechanisms related to ribosome synthesis remain elusive. The yeast nucleolar protein Nop2p is essential for cell viability and growth in Saccharomyces cerevisiae. In order to dissect its function, conditionally lethal strains were constructed either by placing the open reading frame under the control of a repressible promoter, GAL10, or by mutagenizing the gene. Depletion of, and mutation in, Nop2p caused a significant reduction in the levels of the 25S rRNA and the 60S ribosomal subunit. Pulse-chase analysis of the processing of pre-rRNA in these conditionally lethal strains revealed that the processing was significantly delayed with the accumulation of 35S, 32S and 27S pre-rRNAs and with a significant reduction in the formation of 25S and 5.8S rRNAs. Northern blotting analysis indicated that both 27SA and 27SB pre-rRNAs accumulated during Nop2p depletion. Since protein alignment vi

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analysis suggests that the protein may have methylase activity, the role of Nop2p in methylation of rRNAs was investigated by RNase protection assay. Depletion of Nop2p caused a statistically significant reduction in methylation at a conserved site in 27S prerRNA. Mutational analysis of the protein revealed that the multiple missense mutations in the gene were responsible for the temperature-sensitive or formamide-sensitive phenotypes. The mutations in the six nop2 alleles caused a defect in the processing of pre-rRNA to 25S rRNA. The 27S pre-rRNA, which accumulated in the other four nop2 alleles, was rapidly degraded in nop2-4 and nop2-5 alleles. These findings clearly indicate that Nop2p is required for the synthesis of 60S ribosomal subunit and processing of 27S pre-rRNA to 25S and 5.8S rRNAs. This study also helps elucidate the molecular mechanisms related to ribosome synthesis. VII

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INTRODUCTION The Nucleolus and Ribosome Synthesis The nucleolus is a membrane free organelle within the nucleus. Morphologically, three substructures can be recognized in the interphase nucleoli of higher eukaryotic cells: fibrillar centers (FCs), dense fibrillar component (DFC) and granular component (GC). (Warner, 1990; Scheer et al., 1993; Scheer and Weisenberger, 1994). Since FCs contain rDNA and the enzymes essential for its transcription (RNA polymerase I, DNA topoisomerase I and transcription factors), it is believed that the primary rRNA transcript is generated in FCs. Fibrillarin, a nucleolar protein, was found to be localized in this substructure. Since fibrillarin is involved in pre-rRNA processing, DFC is likely the site of early steps in pre-rRNA processing. The late events of maturation of ribosome precursor particles occur in GC. The yeast nucleolus appears as a single, crescent-shaped region occupying more than 30% of the nucleus (Woolford, 1991). Although there are no obviously distinct substructures in the yeast nucleolus, ribosome synthesis is also the main function of yeast nucleolus (Warner, 1989; Melese and Xue, 1995). The yeast ribosome is a large complex of proteins and RNAs, which consists of 40S and 60S ribosomal subunits. The 40S subunit contains about 32 different proteins and an 18S rRNA, while the 60S subunit contains about 45 proteins and three rRNAs: 5S, 5.8S and 25S (28S in higher eukaryotes) rRNAs (Woolford, 1991). Since the ribosome is a protein-synthesizing machine, synthesis of the ribosome itself is essential for cell growth and maintenance. The nucleolus is the center for ribosome synthesis (Table 1). In the nucleolus, repeated rDNA genes are transcribed by RNA polymerase I into a single large

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Table 1. Main Events in Ribosome Synthesis Event Location Comments rDNA transcription 5S rDNA transcription Pre-rRNA processing Modification of rRNAs Transcription of ribosomal protein (r-protein) genes Processing of mRNAs of r-proteins Translation of r-proteins Modification of r-proteins Import of r-proteins Assembly Nucleolus Nucleolus Nucleolus (except 20Stol8SrRNA in the cytoplasm) Nucleolus Nucleus Nucleus Cytoplasm Cytoplasm or nucleolus? Cytoplasm to nucleolus Nucleolus Transcribed by RNA polymerase I into a large single precursor containing 18S, 5.8S and 25S rRNA sequences. Transcribed by RNA polymerase III; no processing or modification has been found. Involving a series of cleavages; simultaneous with the modification and assembly. Including base methylation, ribose methylation, and pseudouridylation during the transcription. RNA polymerase II. Including capping, splicing and polyadenylation. Including methylation, phosphorylation and acetylation. Involving nuclear localization signal (NLS) or "piggy-back import" mechanism. Involving protein-protein and protein-RNA interaction. Export of ribosome subunits Nucleolus to cytoplasm

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35S (45S in higher eukaryotes) precursor, which is then modified and cleaved into three mature rRNAs: 18S, 5.8S and 25S rRNAs. 5S rRNA is generated by RNA polymerase III. Coupled with this maturation process, ribosomal proteins, imported from the cytoplasm, are assembled with pre-rRNAs and 5S rRNA to form functional 40S and 60S ribosomal subunits. The ribosomal subunits are then exported to the cytoplasm for protein synthesis (Warner, 1989; Woolford and Warner, 1991; Sollner-Webb et al., 1996). Processing of Pre-rRNA The single, large pre-rRNA contains 18S, 5.8S and 25S rRNA sequences, which are flanked by 5' and 3' external transcribed spacer (ETS), and separated by internal transcribed spacer (ITS) 1 and 2 (Figure 1). Release of the rRNAs from the precursor is, therefore, essential for ribosome synthesis. Processing of pre-rRNA into mature rRNAs involves multiple endonucleolytic and exonucleolytic cleavages. Earlier studies of prerRNA processing in unicellular organisms (Saccharomyces cerevisiae), plants (Phaseolus vulgaris), insects (Drosophila), amphibia (Xenopus laevis) and several mammals suggest that a common processing pathway is generally followed (Crouch, 1984; Hadjiolov, 1985; Tollervey, 1987; Gerbi et al., 1990; Eichler and Craig, 1994; Sollner-Webb et al., 1996). In yeast, the processing starts at 5' ETS (Figure 1). Cleavage at An generates a 33S intermediate, which is then converted to 32S with the processing at A\ site. These early processing steps generate the 5' end of 18S rRNA. Cleavage at A2 within ITS1 separates the processing pathway into two halves. One is conversion of 20S into 18S rRNA, which occurs in the cytoplasm. Another is to form 5.8S and 25S rRNAs. When the processing of ITS 1 and 3' ETS is completed, 27SA becomes 27SB. 27SB is the common precursor for 5.8S and 25S rRNAs. Cleavage at C2 site within ITS2 generates two intermediates. One contains ITS2 and 25S rRNA sequence. With the subsequent processing at C\, the mature 25S rRNA is formed. Another is 7S intermediate, the direct precursor for 5.8S

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rRNA. With exonucleolytic "chewing" from C2 to E site, 7S is converted to 5.8S rRNA (Tollervey, 1987). A recent study revealed that there are two versions of 5.8S rRNA in the wild type cells. The larger 5.8S (5.8SrJ containing several extra nucleotides at 5' end is a minor version (Henry et al., 1994). Modification of rRNAs and Ribosomal Proteins Methylation of rRNAs All eukaryotic rRNAs are modified by methylation, though the number of the methylations varies among different organisms. There are a total of 1 16 methylations in human rRNAs, while only 65 methylations in yeast rRNAs (Maden et al., 1995). Nevertheless, from yeast to human, a great majority (80-90%) of the methylations are 2'O-methylations. No consensus sequence for rRNA methylation has been found. However, almost all eukaryotic rRNA methylations are in the conserved core regions of rRNA: the regions with main secondary structure features conserved from archaea to bacteria to eukaryotes (Maden, 1990; Maden, 1996). In yeast, most methyl groups are added soon after transcription at the level of 35S and 32S precursors (early methylation). Very few are added at the level of the late processing intermediates at the level of 27S and 20S intermediates (late methylation). Among the methylations identified in the 25S rRNA sequence, only four were found to be added at the level of 27S level: two copies of mU (base methylations) and UmGm v FUC2922 (2'-0-methylations) (Brand et al., 1977). The function of the methylation remains elusive. Since methyl groups add hydrophobic patches to rRNA and since all methylations are in the conserved core regions, the modification may facilitate rRNA folding or rRNA-protein interactions, and thus may be involved in ribosome synthesis. Some earlier studies found that the methylation of rRNAs was required for pre-rRNA processing and maturation of ribosomes. In cultured

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mammalian cells, methionine deprivation or treatment with ethionine, a chemical inhibitor of methyl transfer, caused a defect in ribosome maturation and pre-rRNA processing (Swann et al., 1975; Caboche and Bachellerie, 1977; Wolf and Schlessinger, 1977). The methylation of rRNA may also be involved in the ribosome function. A higher concentration of methyl groups was found in and around the peptidyl transfer region of the large subunit rRNA, and several modifications in 1 8S rRNA were identified on the platform of the small ribosomal subunit, where mRNA and tRNA binding occurs (Maden, 1995). Demethylation of rRNAs was found to be related to the reduced protein synthesis (Wolf and Schlessinger, 1977). Recent studies of Dimlp (a yeast 18S rRNA methylase) and a yeast temperature-sensitive nopl mutant suggest that the ribosome containing the demethylated rRNAs was defective in its function. However, the demethylated pre-rRNA was still processed and methyl deficient ribosome could still be formed (Tollervey et al., 1993; Lafontaine et al., 1995). These findings suggest that rRNA methylation may be required for ribosome function. Nevertheless, the relationship between methylation and pre-rRNA processing and ribosome maturation has not been clearly understood. Pseudouridylation of rRNAs Eukaryotic rRNAs are also modified by pseudouridylation. The ratio of pseudouridylation to 2'-0-methylations is close to 1:1 in yeast (47:55), amphibian (98:98) and human (95:105) rRNAs (Maden et al., 1995), suggesting that the two types of modifications may be interdependent. Since pseudouridine has more versatile hydrogenbonding properties than uridine, it was speculated that pseudouridine contributes to the recognition events crucial to ribosomal function. However, the exact function of this modification has not been understood.

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Modification of Ribosomal Proteins Ribosomal proteins were found to be modified by methylation, phosphorylation or acetylation (Raue and Planta, 1991; Woolford and Warner, 1991). Several yeast ribosomal proteins belonging to the small subunit (S31 and S2) and to the large subunit (L3, L15, L41, and L42) were found to be methylated (Cannon et al., 1977; Kruiswijk et al., 1978). Since addition of a methyl group increases the local hydrophobic ity, it is possible that methylation of ribosomal proteins may be related to protein-protein association during the assembly. The study of methylation of the ribosomal protein L3 in E.coli supports this possibility. The only modification in L3 is /Y-^-methylglutamine, which is modified by the prmB protein. The E.coli mutant lacking this methylase activity displayed a cold-sensitive phenotype. At 22C, the mutant cells synthesize abnormal ribosomal particles that are very unstable and quickly degraded (Lhoest and Colson, 1977; Muranova et al., 1978; Lhoest and Colson, 1981). However, in eukaryotic cells, such protein methylases have not been identified. The role of methylation of ribosomal proteins in ribosome synthesis is not known. The function of phosphorylation and acetylation of ribosomal proteins is not known. The lack of acetylation (such as in rp39, rp52, rp56 and rp59) or the lack of phosphorylation (such as in S10) did not affect cell growth, suggesting that these modifications may not function in ribosome synthesis (Johnson and Warner, 1987; Woolford and Warner, 1 99 1 ; Wachtler and Stahl, 1993).

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Functions of Nucleolar Proteins Pre-rRNA Processing Non-ribosomal proteins in the nucleolus are thought to play important roles in the synthesis of ribosomes. The number of these nucleolar proteins is believed to be very large (HernandezVerdun, 1991). A number of the nucleolar proteins required for rDNA transcription have been identified (Reeder, 1990). However, the functions of the nucleolar proteins in the other events of ribosome synthesis have just recently become understood. Although the general outline for pre-rRNA processing (Figure 1 ) has been known for years, the enzymes and regulatory factors required for the processing have been largely unknown until recently. Several nucleolar proteins, such as fibrillarin, B23 and nucleolin, were described in high eukaryotic cells years ago (Michalik et al., 1981; Ochs et al., 1985; Caizergues et al., 1987). However, their functions remain unclear, mainly because of technical difficulties in the study of high eukaryotic cells. The yeast system is easily accessible to powerful genetic and molecular biology techniques. Recent studies in the yeast model have generated a large amount of knowledge about the roles of the nucleolar proteins in pre-rRNA processing (Figure 2). Fibrillarin, localized in the DFC of the nucleolus, was first found in Physarum and later identified in mammalian cells (Christensen et al., 1977; Ochs et al., 1985). Since its yeast homologue, the Nopl protein (Noplp), was identified (Aris and Blobel, 1991), the protein function has been extensively studied. Human or Xenopus fibrillarin can complement a yeast nopl mutant (Jansen et al., 1991), suggesting that the protein function is evolutionarily conserved. By genetic manipulation, NOP1 was placed under the control of a repressible promoter. Depletion of this protein caused a progressive impairment of all pre-rRNA processing steps, particularly the pathway leading to 1 8S rRNA (Tollervey et al., 1991). The finding suggests that Noplp is required for the processing in 5' ETS and

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11 oo H W fn oo 00 h oo 00 in g ex en p. m ex ex OO a. CX o CX X) Q ex 2 5 CX a. a, ex u BB a ex o ex o ^ z E z Z Q o2 3 X 00 oo oo m CO Q ex ex 1— 1 <4-H H
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12 ITS1. Further study of the temperature sensitive (ts) mutations in NOP1 gene showed that the five nopl ts alleles affected ribosome synthesis at different steps, including pre-rRNA processing, pre-rRNA modification and ribosome assembly (Tollervey et al., 1993). The Sof 1 protein was isolated by screening for the extragenic suppressors of the ts phenotype caused by incomplete complementation of a nopl disruption by human fibrillarin. Sof lp was found to be associated with Noplp, and depletion of it caused a defect in 18S rRNA production (Jansen et al., 1993), suggesting that Sof lp is required for processing in the 5' ETS and/or ITS1. Another nucleolar protein required for 18S rRNA formation is Garlp, which shares a glycine, arginine-rich domain (GAR domain) with Noplp (Girard et al., 1992). Nop3p, also sharing a GAR domain with Noplp, is required for the conversion of 27SB pre-rRNA to 25S rRNA, and 20S pre-rRNA to 18S rRNA (Russell and Tollervey, 1992). Nop77p was isolated by screening for genes synthetically lethal with nopl -5 ts allele (Berges et al., 1994). The same protein was also identified by another lab at the same time and termed Nop4p (Sun and Woolford, 1994). Nop77p/Nop4p is required for the conversion of 27SA to 27S B pre-rRNA, since no 27SB pre-rRNA was formed in the cells depleted of the protein. This suggests that the protein is required for the processing in 3' ITS1. All these findings indicate that fibrillarin (Noplp) is the center of more than one protein-protein and protein-snoRNAs (see below) complex, which mainly functions in the processing of 5' ETS and ITS1. Several putative ATP-dependent RNA helicases in yeast have been characterized. These proteins contain the evolutionarily conserved DEAD-box. The DRS1 gene was isolated by complementation of cold-sensitive phenotype of drsl. In the drsl mutant, processing of 27S pre-rRNA to 25S rRNA was impaired (Ripmaster et al., 1992). Another yeast putative RNA helicase is Spb4p, which is also involved in the processing of 27S pre-rRNA to 25S rRNA (Sachs and Davis, 1990). However, it is not clear if Drslp and Spb4p are involved at the step of 27SA to 27SB pre-rRNA, or at the step of 27SB to 25S rRNA. Recently, another putative RNA helicase, Dbp3p, was isolated. This protein

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13 was found to be required for processing at A3 site in ITS 1 The gene is not essential. However, disruption of it caused a delay in the processing of 27SA to 27SB pre-rRNA, and thus a significant reduction in the formation of 25S rRNA (Weaver et al., 1997). The mechanism by which these putative RNA helicases are involved in pre-rRNA processing is not clear. It was speculated that RNA helicase may modulate the secondary structure of pre-rRNA for recruiting the RNase MRP complex (see below) to A3 site (Weaver et al., 1997). Several other proteins were found to be involved in processing ITS 1 In rrpl ts mutant, 27S pre-rRNA was rapidly degraded and the formation of 25S rRNA was reduced (Fabian and Hopper, 1987). It is not clear if 27SB pre-rRNA was formed or not. RNase MRP, an endoribonuclease, is required for processing at A3 site in ITS1. Mutation in its protein component (ts popl-1) or in its RNA component (rrp2-l) caused a defect in the cleavage at the A3 site (Lygerou et al., 1994; Morrissey and Tollervey, 1995). Xrnlp and Ratlp were found to have 5' to 3' exonuclease activity. Mutation in RATI gene caused a reduction in the formation of the short form of yeast 5.8S rRNA (Amberg et al., 1992). Mutation in XRNJ gene resulted in the accumulation of the excised 5' region of ITS 1, between the 3 end of 18S rRNA and A2 site (Stevens et al., 1991). Ratlp and Xrnlp were found to digest from site A3 to Bi (s) to form the 5' end of 27SB (s) pre-rRNA (Henry et al., 1994). Yeast RNT1, a homologue of bacterial RNase III, was found to encode a doublestrand endoribonuclease, which is required for processing both the 5' and the 3' ETS. This endonuclease activity was reproduced in vitro in the absence of snoRNA, suggesting that snoRNA had no catalytic role in this situation (Elela et al., 1996). Nsrlp, a yeast homologue of nucleolin, is required to form 20S pre-rRNA, a direct precursor for 18S rRNA. The cells lacking NSR1 were viable but had a severe growth defect and an near absence of 20S pre-rRNA (Lee et al., 1992).

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14 Since pre-rRNA processing involves multiple orderly cleavage steps (Figure 1), it is not surprising that so many nucleolar proteins are required for its processing. So far, no yeast nucleolar protein has been clearly shown to be required for ITS2 processing. The detailed mechanisms for the other processing steps have still not been completely understood. Therefore, much more remains to be learned about the functions of the nucleolar proteins in pre-rRNA processing. Modification of rRNAs It has been known for many years that all eukaryotic rRNAs are modified by methylation (Maden, 1990). However, only recently have several putative eukaryotic rRNA methylases and the regulatory factors been characterized. Noplp was found to be required for rRNA methylation (Tollervey et al., 1993). Since Noplp is associated with a large number of the nucleolar proteins and snoRNAs (see below) and since it was found to be involved in every major step of ribosome synthesis (Tollervey et al., 1993), it is most possible the Noplp is a multi-functional snoRNP protein. Human proliferation-associated nucleolar protein pi 20 was found to contain a sequence conforming to an S-adenosylmethionine (SAM) binding domain, which is present in a variety of the known methylases (Koonin, 1994). However, it has not been shown that pi 20 possesses rRNA methylase activity. Segal and Eichler identified and partially purified a novel nucleolar methylase from the crude mouse nucleolar extracts. This protein was found to methylate the 28S rRNA transcript at a unique site in vitro (Segal and Eichler, 1991). However, the gene has not been isolated. Recently, two yeast rRNA methylases have been identified. Pet56 protein was found to be required for ribose methylation at a universally conserved nucleotide in the peptidyl transferase center of the mitochondrial large ribosomal RNA (2 IS rRNA). This was confirmed by the result from the in vitro methylation reaction. The reduction in this

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15 protein function caused a defect in the formation of large subunit of mitochondrial ribosome (Sirum-Connolly and Mason, 1993), which suggests that the rRNA methylation may be required for ribosome assembly or stability. Yeast Dim 1 protein was cloned by complementation of the kasugamycin sensitivity of the E.coli ksg A mutant (Lafontaine et al., 1994). KsgA is a 16S rRNA dimethylase in E.coli (van Buul and van Knippenberg, 1985). The study of Dimlp function showed that this protein also had dimethylase activity in yeast. This protein was found to be required for the methylation at a specific site on 18S rRNA. However, such methylation was not required for the formation of 1 8S rRNA, though Dimlp itself was essential for cell viability (Lafontaine et al., 1995). Considering that all eukaryotic rRNA are methylated at a large number of sites, more rRNA methylases probably remain to be identified, and the significance of rRNA methylation in ribosome synthesis remains to be studied. Modification of Proteins Ribosomal proteins are covalently modified (Raue and Planta, 1991; Woolford and Warner, 1991). However, it is not known if such modification occurs in the cytoplasm and/or the nucleolus. Since all of the ribosomal proteins are imported to the nucleolus to assemble with rRNAs into ribosome subunits, it is possible that the nucleolar proteins may modify some of the ribosomal proteins to facilitate the assembly. Some nucleolar proteins, such as nucleolin, fibrillarin, and Ssblp, were found to be methylated (Lischwe, 1990). All of these nucleolar proteins contain the glycine, arginine-rich domain (GAR domain) interspersed with phenylalanine, which is thought to be recognized by a group of protein methylases (Lischwe, 1990). The methyl groups were found to be added to the arginine residues as N&, A^-dimethylarginine (Lischwe et al., 1982; Lischwe et al., 1985). The exact function of the arginine methylation is not known. Since the GAR domain was found to have a high affinity for nucleic acid (Cobianchi et al.,

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16 1988), it was believed that such modification is related to nucleic acid binding. Since the methyl group increases the local hydrophobicity, a role of the modification in proteinprotein interaction is also possible (Lischwe, 1990). However, the methylases required for the modification have not been identified. These nucleolar proteins are mainly localized in the nucleolus (Lischwe et al., 1985; Lapeyre et al., 1987; Clark et al., 1990), Therefore, it is possible that other nucleolar proteins with protein methylase activity may modify these proteins. Assembly of Ribosomes Ribosome assembly involves association of approximately 70-80 ribosomal proteins and 4 rRNAs through protein-protein and protein-rRNA interactions. In E. coli, ribosomal subunits can be successfully assembled in vitro with only rRNA and ribosomal proteins (Nomura, 1973). However, such in vitro assembly has not been successful for eukaryotic ribosomes, suggesting that some cofactors are required for proper assembly. In vivo labeling in yeast showed that ribosomal precursor particles contained not only ribosomal proteins, but also a number of non-ribosomal proteins (Warner, 1971), suggesting that nucleolar proteins may participate in the assembly process. Nucleolin and B23 in higher eukaryotes were found to be associated with preribosomal particles in the granular component of the nucleolus (Spector et al., 1984; Biggiogera et al., 1989), where pre-ribosomal subunits occur. Since both proteins contain acidic domains (Spector et al., 1984; Lapeyre et al., 1987), it was speculated that the proteins may function by facilitating assembly of the many basic ribosomal proteins with rRNAs (Woolford, 1991). Several other nucleolar proteins also contain an acidic domain, including Noppl40, Nsrlp, Drslp, and Nop77p/Nop4p (Melese and Xue, 1995). It was believed that the acidic domain in these proteins may interact with the highly basic NLS or NLS-like sequences in many ribosomal proteins and thus to facilitate ribosome assembly

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17 (Xue and Melese, 1994; Melese and Xue, 1995). The glycine, arginine-rich domain (GAR domain) is present in many nucleolar proteins, including fibrillarin (Noplp), Nsrlp, Ssblp, nucleolin, Garlp, and Nop3p (Schimmang et al., 1989; Lee et al., 1991; Jong et al., 1987; Lapeyre et al., 1987; Girard et al., 1992; Russell and Tollervey, 1992). It is possible that GAR domain in these proteins may promote proper assembly of ribosome by interacting with some acidic ribosomal proteins, such as yeast AO, Al, L44, L44' and L45 (Woolford and Warner, 1991). Since ribosome assembly involves interaction among ribosomal proteins and rRNAs, modification of rRNA by the nucleolar proteins may play a role in assembly. One piece of evidence is from the study of a yeast ts nopl.3 mutant (Tollervey et al., 1993). In this mutant, the methylation of pre-rRNA was strongly inhibited at 36C, but the demethylated pre-rRNA was still processed. However, the lethal phenotype caused by this allele at 36C suggests that the synthesized ribosomes were non-functional. Functions of SnoRNAs Small nucleolar RNAs (snoRNAs) have been identified in a variety of eukaryotic cells (Fournier and Maxwell, 1993). The number of snoRNAs is very large. In yeast, more than 30 snoRNAs have been isolated (Balakin et al., 1996). Some snoRNAs have been shown to be required for pre-rRNA processing. Depletion of either U3, U14, or snR30 caused a severe defect in the formation of 18S rRNA (Li et al., 1990; Hughes and Ares, 1991; Morrissey and Tollervey, 1993), suggesting that these snoRNAs function in the processing of 5' ETS and/or ITS 1 (Fournier and Maxwell, 1993). SnRlO, though not essential, participates in the processing of 35S pre-rRNA (Tollervey, 1987). However, other snoRNAs have not been found to be essential for cell viability and pre-rRNA processing (Sollner-Webb et al., 1996).

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18 Recently, it has been found that a family of snoRNAs are required for the site selection of rRNA 2'-0-methylation (Maden, 1996;Tollervey, 1996). There are two important features about these snoRNA. First, they contain two evolutionary conserved motifs, boxes C and D, which are believed to be protein binding sites. Second, the snoRNAs exhibit long (14-17 nucleotide) regions of perfect complementarity to pre-rRNA (Maden, 1996; Tollervey, 1996). Mutations altering the sequence of the snoRNA or the spacing between box D and the region of base paring is sufficient to change the specificity of methylation (Cavaille et al., 1996; Kiss-Laszlo et al., 1996). These studies suggest that the 2'-0-methyltransferase may consist of a protein component, which catalyzes transfer of a methyl group, and an snoRNA component, which selects the modification site. Interaction of Nucleolar Proteins and SnoRNAs Several pieces of evidence indicate that nucleolar proteins and snoRNAs may functionally and physically interact. First, depletion of Noplp, Garlp, Sof lp and Nsrlp cause phenotypes that are strikingly similar to phenotypes caused by depletion of U3, U14, and snR30, which suggests that these proteins and RNAs function at the same or similar steps of pre-rRNA processing (Li et al., 1990; Hughes and Ares, 1991; Tollervey et al., 1991; Girard et al., 1992; Lee et al., 1992; Jansen et al., 1993; Morrissey and Tollervey, 1993). Second, immunoprecipitation studies indicate that the nucleolar proteins are physically associated with snoRNAs. At least 1 1 yeast snoRNAs can be precipitated by antibodies to Noplp (Schimmang et al., 1989). Garlp and Sof lp were found to be associated with snRlO and snR30 (Girard et al., 1992), and with U3 (Jansen et al., 1993), respectively. Finally, a majority of the nucleolar proteins identified so far ( such as Noplp, Nop3p, Garlp, Nsrlp, Ssblp, fibrillarin and nucleolin) contain the consensus motifs of RNA recognition motif (RRM) or/and GAR domain (Ochs et al., 1985; Jong et al., 1987; Lapeyre et al., 1987; Schimmang et al., 1989; Lee et al., 1991; Girard et al., 1992; Russell

PAGE 26

19 and Tollervey, 1992). The RRM consists of an amino acid sequence of -80 residues conserved among the members of a family of RNA-binding proteins (Kenan et al., 1991). The aromatic and basic residues in the RRM have been implicated in nucleic acid interaction (Kenan et al., 1991). The GAR domain consists of repeating units of RGGFRGG (Russell and Tollervey, 1992). Study of the GAR domain of vertebrate nucleolin suggests that the domain is involved in non-specific protein-RNA association (Ghisolfi et al., 1992). On the other hand, all fibrillarin (Noplp)-associated snoRNAs contain two consensus sequences, boxes C and D, which may be the binding sites for common proteins (SollnerWebb et al., 1996). Another family of snoRNAs have consensus AC A triplet positioned 3 nucleotides before the 3' end of the RNA. All members of the yeast AC A family are associated with Garlp (Balakin et al., 1996). Based on the above evidence, it was proposed that nucleolar proteins are associated with snoRNAs to form snoRNP complex, which may be involved in the processing and modification of pre-rRNA, ribosome assembly and transport (Fournier and Maxwell, 1993). Nop2p is a yeast homologue of human nucleolar protein pi 20, which is a marker for cell proliferation (de Beus et al., 1994; Fonagy et al., 1993). Nop2p is different from most of the known nucleolar proteins in that it does not contain either RRM or GAR domain (de Beus et al., 1994). However, like pl20, Nop2p contains a putative Sadenosyl-methionine (SAM) binding domain, which is conserved among methylases. In this study, by genetic manipulation, the conditionally lethal strains were constructed and a deficit in Nop2p function was investigated at the restrictive conditions. The relationship between Nop2p and methylation was analyzed. By using immunological and genetic approaches, possible functional interaction between Nop2p and other macromolecules was explored.

PAGE 27

METHODS AND MATERIALS Strains, Media, and Microbiological Methods Saccharomyces cerevisiae strains used in this study are listed in Table 2. Yeast cells were grown at 30C or as indicated. The complex media were either YPD (1% yeast extract, 2% peptone, and 2% dextrose) or YPGal (1% yeast extract, 2% peptone, and 2% galactose). The synthetic minimal media were either SD (0.67% yeast nitrogen base and 2% dextrose) or SGal (0.67% yeast nitrogen base and 2% galactose), supplemented with appropriate nutrients (Sherman, 1991). Methionine was always omitted from the synthetic media in this study. For [ 3 H]uracil labeling experiment, the concentration of uracil in the synthetic media was reduced to one third the normal amount (Sherman, 1991). For plasmid shuffling, 1 mg/ml of 5-fluoroorotic acid (5-FOA) (PCR Incorporated) was added to either SD or SGal medium (Boeke et al., 1987). For screening for formamide-sensitive or ethanol-sensitive strains, 3% formamide (Aguilera, 1994) or 6% ethanol (Aguilera and Benitez, 1986) was added into SD medium, respectively. YPD containing low phosphorus (LPYPD) was prepared as described previously (Reid and Schatz, 1982). Briefly, YP (1% yeast extract, 2% peptone) was treated with 10 mM MgS04 and 1% NH 4 OH at room temperature (RT, ~25C) for 30 minutes, and filtered twice through Whatman 3MM paper before adding 2% dextrose. For screening for thermosensitive strains, yeast cells were replica plated and grown at permissive temperature (25C) and non-permissive temperature (14Cor37C). Yeast genetic manipulation was done according to previously described methods (Guthrie and Fink, 1991) with minor changes. Yeast cells were first grown at 30C on 20

PAGE 28

21 selective SD plates for 2-4 days. A small dab of cells was patched onto the pre-sporulation plate (0.8% yeast extract, 0.3% peptone, 10% dextrose, and 1.5% agar.) and grown at 30C for -24 hours. Cells from pre-sporulation were then made a patch onto the sporulation plate (1% KOAc, 0.1% yeast extract, 0.05% dextrose, 1.5% agar, and with one fourth the normal amount of appropriate nutrients). The spore wall of asci was digested with 6% glusulase (DuPont) at room temperature for 6 minutes. The digested asci were then spread onto a YPD plate, and spores were separated by micromanipulation. The spores were germinated and grown on YPD plates. DNA Methods All of the plasmids used in this study (Table 3) were maintained and amplified in E. coli DH5oc in LB medium (0.5% Bacto-yeast extract, 1% Bacto-tryptone, 0.5% NaCl, pH 7.5, and 1.5% agar for plates only) plus 100 Hg/ml ampicillin. Plasmids were introduced into E. coli DH5a by using the RbCl method (Hanahan, 1983). DNA was transformed into yeast cells by using the lithium acetate method (Ito et al., 1983). Yeast genomic DNA was prepared as described (Wack et al., 1994). An overnight culture of yeast in 2.5 ml of YPD (~5 OD600 units) was collected and resuspended in TE buffer (10 mM TrisHCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0) with 0.5 M sorbitol. Cell wall was digested with -400 units of Lyticase (Sigma) and 5 |iM dithiothreitol (DTT) at RT for 30 minutes. Spheroplasts were broken in 200 ul of 50 mM TrisHCl (pH 8.0), 20 mM EDTA and 1 .25% sodium dodecyl sulfate (SDS) at 65C for 20 minutes. After adding 100 \i\ of 3 M potassium acetate (pH 5.0), the mixture was centrifuged at top speed. DNA in the supernatant was precipitated with ethanol. RNA in the pellet was eliminated by enzymatic digestion with 2 Hg/ml RNase A. DNA was again precipitated by ethanol.

PAGE 29

22 Recovery of plasmid from yeast was done according to the method of Strathern and Higgins (Strathern and Higgins, 1991). Briefly, an overnight culture in 2 mis of YPD was collected and resuspended in 100 u.1 of TENS buffer (100 mM NaCl, 10 mM TrisHCl, pH 8.0, 1 mM EDTA, and 0.1% SDS). The cells were broken by vortexing with 0.25 g of glass beads for one minute. The cell lysate was treated with phenol. The crude preparation containing plasmid DNA was obtained by treating the aqueous layer with a Geneclean kit (Bio 101). Using a gene pulser (BioRad), the recovered plasmid DNA (in 3 |il) was introduced into E. coli. DH5cc. Electro-competent E. Coli. DH5a cells were prepared as described below. An overnight culture of E. coli. DH5a in 5 ml of 2X YT medium ( 1 .6% Bacto-tryptone, 1 % Bacto-yeast extracts and 0.5% NaCl) was diluted into 500 ml of 2X YT and grown at 37C for 2-3 hours. The cells were collected and washed twice with ice cold water. The cells were washed with ice cold 10% glycerol. Finally, the cells were resuspended in 1 ml of ice cold 10% glycerol. Aliquots of the cells were stored at -80C. The restriction enzymes used in this study were purchased from New England Biolabs. Restriction digestions were performed according to the manufacturer's instructions. DNA fragments were isolated as described below. After separation of DNA fragments on 0.8% agarose gels, a piece of Whatman GF/C filter paper on a piece of dialysis tubing membrane was inserted facing the band of interest. After electrophoresis for 20-30 minutes, the GF/C paper was removed, and the DNA on the paper was obtained by brief centrifugation. After adding an equal volume of phenol, the mixture was added onto a sephadex G-50 spin column. The DNA was recovered by spinning the column in IEC centrifuge at 1,000 rpm (~250g) for 10 minutes. Southern blotting was done as described (Ausubel et al., 1993). Briefly, after restriction digestion, the DNA samples were separated on a 0.8% agarose gel. The gel was treated sequentially with 0.25 M HC1 for depurination, with denaturation buffer ( 1 .5 M

PAGE 30

23 NaCl, 0.5 M NaOH), and with neutralization buffer (3 M NaCl, 0.5 M Tris, pH7.0). Transfer of DNA from the gel to a piece of nitrocellulose paper was done with upward capillary transfer in 20X SSC buffer (3 M NaCl, 0.3 M Na 3 Citrate-2H 2 0, pH 7.0). The nitrocellulose paper was then baked at 70C for 2 hours. Probes were labeled with [a32 P]dCTP by random oligonucleotide priming. Manual DNA sequencing was done by using the dideoxy method (Ausubel et al., 1993). The double-strand template carried on a plasmid was denatured in 1 M NaOH at 37C for 5 minutes. The DNA with 2.0 pmol of a primer in 5X sequence reaction buffer (200 mM TrisHCl, pH 7.5, 100 mM MgCl 2 250 mM NaCl) was incubated at 65C for 2 minutes, and slowly cooled to 30C. The annealed template-primer was mixed with 0. 1 M DTT, 1.5 M dCTP, 1.5 M dGTP, 1.5 M dTTP, 5 uCi [cc-35S]dATP, and 3 units of Sequenase 2.0 (Amersham). The mixture was incubated at 20C for 2-5 minutes. For each labeling reaction, 2.5 ill of ddATP, ddCTP, ddGTP, or ddTTP was added to four wells in a microwell culture dish, and incubated at 42C. The labeling mixture was added to each well and incubated at 42C for 2-5 minutes. Then 4 jlxI of stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol FF) was added to each well. After denaturation with steam for 2 minutes, the mixture in each well was separated on 8 M urea-6% polyacrylamide gel, and detected by autoradiography. The probes used for sequencing are listed as followings: P3, 4, 5, 6, 7, 8, 9, 10, 12, 21, 23, 24, 25, 26, 27, 28, 29 (see Table 4). Construction of GAL-NOP2 Strain Gene disruption was performed according to the method described by Rothstein (Rothstein, 1991). A diploid strain YPH501 was transformed with aNOP2::HIS3 disruption fragment (Figure 3). Correct integration of this fragment into one NOP2 locus in the His+ transformants (YSB12) was confirmed by Southern blotting (not shown).

PAGE 31

Figure 3. An intact copy of NOP2 gene is present in the cells from all of the viable spores. The restriction maps of NOP2, nop2::HIS3 fragment and the probe are as shown. The genomic DNA from the cells germinated from the two spores of one ascus (lane 1 and 2) and the four spores of another ascus (lane 3, 4, 5, and 6) was digested with HindlU and analyzed with Southern blotting.

PAGE 32

25 Xhol EcoRI Hpal BamHI 2 1290 1966 2408 Bgill 5856 p^4vH 1 | 1 I j \ N0P2 2681-4537 | \>'/^] EcoRI Hindlll Clal EcoRI •0 8 kb 954 1693 2630 H B I I IHindlll EcoRI Sad 4393 5012 6 kb H E HIS3 -*/ 3.4 Kb2.2 Kb Probe B 12 3 4 5 6

PAGE 33

Figure 4. Nop2p is expressed in the cells from all of the viable spores. YBH9 and YSB12 are the diploid strains with one NOP2 locus disrupted and replaced by LEU2 or HIS3, respectively. Total cellular proteins were extracted from the cells germinated from the four spores (a, b, c, and d) of the same ascus, separated on a SDSpolyacrylamide gel, and analyzed by Western blotting. The position of Nop2p is indicated. L: the wild-type haploid L4717. The band with a low molecular weight may be non-specific.

PAGE 34

27 Nop2p YBH9 (nop2::LEU2) YSB1 2 (nop2::HIS3) abed abcdL -,a-*MMn-w_ *-—

PAGE 35

Figure 5. Disruption of NOP2 gene in a haploid L4717 strain. (A) The restriction maps of NOP2, nop2::LEU2 fragment and the probe. (B) The nop2::LEU2 strain, YBH3, was dependent upon the plasmid-carried NOP2 gene for viability. Serial dilutions of L4717, YBH12 and YBH3 were grown on SD medium with or without 5FOA. YBH12 was constructed by introducing pJPA40 (NOP2, URA3) into L4717. YBH3 was derived from the disruption of the NOP2 locus in YBH12 by nop2::LEU2 fragment. (C) Disruption of the NOP2 locus was confirmed by Southern analysis. Genomic DNA from L4717 (lane 1), and two NOP2 isolates (lane 2 and 3) and nop::LEU2 isolates (lane 4: YBH3, and lane 5: YBH4) carrying pJPA40 plasmid were digested with EcoRl and analyzed with Southern blotting. The 7.9-kb band corresponds to the linearized pJPA40 plasmid.

PAGE 36

29 Xbol 2 EcoRI Hindu -0 8 kb 954 EcoRI 1290 I Hpal 1966 I I Clal 1693 BamHI 2408 _l NOP2 2681-4537 Bglll 5856 EcoRI 2630 rn Hind HI EcoRI Sad 4393 5012 6 kb LEU2 -*J Probe + 5-FOA 3 4 5 L4717*• • W> vi YBH12^ • YBH37.9 L4717YBH12 YBH3"5-FOA • 9 O $ • O • € • • • -.2.2 -2.1 -1.3

PAGE 37

30 However, tetrad dissection revealed that all of the four spores from the same tetrad were viable with two His" and two His + spores (not shown). To see if Nop2p was required for the viability of the cells from the spores, Western analysis with an affinity-purified polyclonal antibody APpAb3 to Nop2p was done (Figure 4, YSB 12). Nop2p was detected in the cells germinated not only from His" spores (lanes c and d) but also from His + spores (lanes a and b) of the same tetrad (Figure 4). Genomic DNA was digested with HindlU and analyzed with Southern blotting (Figure 3). The His" cells from the two spores exhibited a 3.4-kb band, but no 2.2-kb band (Figure 3, lanes 5 and 6), indicating the cells contained only one intact copy of NOP2. However, The His+ cells from another two spores showed not only 2.2-kb band but also 3.4-kb band (Figure 3, lane 3 and 4), suggesting that the cells contained copies of both the disrupted and the intact NOP2. Considering that this might be a strain-specific phenomenon, several other diploid strains (STX466, 9933D, YSB1 1 and YJA501) were tested, but with similar results (not shown). Disruption with NOP2::LEU2 fragment also showed the same result (Figure 4, YBH9). To avoid this sporulation-related problem, a disruption of the gene was made in a haploid strain in the presence of a plasmid-carried NOP2 gene. Part of the 5' non-coding region and 92% of NOP2 open reading frame (ORF) carried on pJPA40 plasmid was replaced by a BamHUHindm fragment containing the LEU2 gene, which was isolated from plasmid pJJ282. The NOP2::LEU2 disruption fragment (Figure 5A) was then linearized with XhoVSacl digestion. The wild type haploid strain L4717 cells were first transformed with NOP2 gene carried on the pJP40 plasmid (URA3 marker). The Ura+ transformant (YBH12) was viable in the presence of 5-FOA (Figure 5B), suggesting that the cells could survive by losing the URA3 plasmid. The YBH12 cells were then transformed with NOP2::LEU2 fragment (Figure 5A), which, if correctly integrated, should replace 92% of NOP2 ORF and part of the 5 '-non-coding region with LEU2 gene. One of Leu+/Ura+ transformants (YBH3) was dead in the presence of 5-FOA, suggesting that NOP2 gene had been disrupted (Figure 5B). To further confirm the correct integration

PAGE 38

31 of NOP2::LEU2 fragment at the NOP2 locus, genomic DNA from YBH3 and several other Leu + /Ura + isolates was digested with EcoRl, and analyzed by Southern blotting (Figure 5C). In YBH3 (lane 4) and another isolate (lane 5), a 1.3-kb band was lost, but an extra 2.2-kb band was observed, indicating NOP2 gene was disrupted and replaced with LEU2 gene. To generate a conditionally lethal nop2 allele, the NOP2 ORF was placed under the control of a repressible GAL10 promoter. First, a BamHUXhol fragment containing the NOP2 ORF was isolated from pJPA20 plasmid, and subcloned into plasmid pRD53. The SacVXhol fragment containing GAL-NOP2 was excised from this subcloned plasmid (pBH46), and cloned into pRS314 plasmid. The GAL-NOP2 strain (YBH5) was constructed by using plasmid shuffling technique (Sikorski and Boeke, 1991). The pBH47 plasmid was transformed into YBH3 strain. To replace the original plasmid (pJP40) with the second one (pBH47), the Trp+/Ura + transformants were replica plated onto SGal plate containing 5-FOA. One of the colonies formed on the plate, YBH5, was used for the depletion analysis. Cell Growth Curve and Viability Test YBH5 cells were first grown at 30C in YPGal liquid medium until mid-log phase. The cells were then collected, washed and resuspended in water. Equal volume of the cells was transferred into YPD and fresh YPGal media, respectively. At different time points, cell densities were measured by using a spectrophotometer (Beckman) at A6ooTo keep the cells growing in mid-log phase, the cells were diluted into the fresh media whenever cell density reached 0.6-0.8 OD600 unit. The six nop2 temperature-sensitive strains were grown at 25C in YPD, and then shifted to 37C. Cell densities were measured as mentioned above. To test the cell viability after Nop2p depletion, the cells grown in YPD or YPGal medium were collected at the

PAGE 39

32 different time points, diluted to 0.001 OD600 unit and then spread 20 |il (about 160 cells) onto YPGal plates. After incubated at 30C for 2 days, the number of the colonies formed on the plate was counted (Table 5). Isolation of nop2 Conditionally Lethal Alleles In vitro mutagenesis of the NOP2 gene. A low fidelity polymerase chain reaction (PCR) method (Muhlrad et al., 1992) was used to generate random mutations in NOP2 gene. A Kpnl fragment containing NOP2 gene was cut out from pJPA29 plasmid and used as the template. Two oligonucleotides were used as the primers: P19A, complementary to the 5' end; and P16, complementary to the 3' end of the gene. Each of four PCR reactions was set up as followings: 20 ng DNA template, IX reaction buffer (50 mM Tris-HCl, pH 9.0, 50 mM NaCl, and 10 mM MgCl 2 ), 0.4 uM of each primer, 0. 1 mM each of three dNTPs with another dNTP at 0.025 mM, 1 .25 units of Taq DNA polymerase (Promega), with or without 0. 15 mM MnCl2. Each reaction with a total volume of 25 \i\ was overlaid with 100 u.1 of mineral oil, and run in a Thermocycler (Coy) for 30 cycles (for each cycle: denaturation at 94C for 90 seconds; annealing at 55C for 120 seconds; and extension at 72C for 180 seconds). The PCR products from the four reactions were then pooled and gel-purified. In this study, it was found that the majority of nop2 alleles generated in the presence of 0.15 mM MnCl 2 were non-functional even at the permissive conditions, suggesting that Mn 2+ caused a much higher error frequency in the PCR reaction. Preparation of the gapped plasmid. pBH49 plasmid was cut with AatlUSnaBl, and the gapped plasmid was gel-isolated. The sequence homology between the gapped plasmid and the PCR products was -380 bp at the 5' end and -715 bp at the 3' end. It was found that with only 65 bp sequence homology, an efficient recombination still occurred (Muhlrad et al., 1992).

PAGE 40

33 Construction of the stains containing only mutated nop2. Approximately 140 ng of the PCR products and -60 ng of the gapped plasmid were cotransformed into YBH3 by the lithium acetate method. Muhlrad et al. found that homologous recombination between a gapped plasmid and a linear fragment following cotransformation into yeast is efficient (Muhlrad et al., 1992), so that in vitro ligation is not necessary. Plasmid shuffling was performed as described above. As controls, the gapped plasmid or the PCR products alone were also transformed into YBH3. Only 5-10 colonies/plate formed with the gapped plasmid alone, while no colony formed with the PCR products alone. Isolation of conditionally lethal strains. Colonies formed in the presence of 5-FOA were replica plated onto SD medium, and incubated at 14C, 25C and 37C Two other restrictive conditions, 6% ethanol or 3% formamide were also tested at 25C (Aguilera and Benitez, 1986; Aguilera, 1994). Recovery of the plasmids. The plasmid DNA was recovered from the yeast cells, amplified in E, coli DH5a, and verified by restriction digestion. The ORF of nop2 alleles was cut out by digestion with EcoRl, gel-isolated, and subcloned into pBH49 plasmid at the same sites. Mapping of the Mutations in the Six nop2 Alleles The six nop2 alleles carried on pRS313 were manually sequenced and the predicted amino acid substitutions were located (see Figure 19). To identify the mutations contributing to temperature-sensitive (ts) phenotype, the multiple point mutations in each allele carried on pRS313 were separated into several groups by restriction digestion with Bsgl, Mfel or Spel. The restriction fragments containing the mutations were gel-isolated and exchanged with the fragments from NOP2. After ligation, the subclones were amplified in E. coli DH5oc. The subcloned plasmids containing one or several groups of

PAGE 41

34 the point mutations were transformed into YBH3 strain. After plasmid shuffling, the phenotypic effect of the mutations contained in each subcloned plasmid was analyzed by incubating the cells under the restrictive condition (37C). The subcloned plasmids were constructed as described in Table 3. SDS-PAGE and Immunoblotting Total cellular proteins were extracted in the presence of trichloroacetic acid, precipitated, and separated on 10.5% polyacrylamide gel by SDS gel electrophoresis (SDSPAGE), and analyzed by immunoblotting with affinity-purified polyclonal anti-Nop2p antibody (APpAb3) (de Beus et al., 1994). A monoclonal antibody (1G1) against the poly (A) binding protein (PablP) (Anderson et al., 1993) was used to probe the same blot. Measurement of Ribosomes and Polysomes The levels of ribosomal subunits, monoribosomes and polyribosomes were measured as described previously (Bairn et al., 1985). Cells in mid-log phase were collected, washed with lysis buffer (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 30 mM MgCl2) containing 100 p.g/ml cycloheximide, and broken by vortexing with acid-washed glass beads. The lysate was centrifuged at 10,000g at 4C for 5 min., and the supernatant was loaded onto 15%-50% sucrose gradient. The gradient was centrifuged at 40,000 rpm (285,000g) for 2.5 h at 4C in SW41Ti rotor (Beckman Instruments, Inc.). After centrifugation, the gradient was analyzed by using an absorbance detector (ISCO UA-5). The polysome curves were traced by using Freehand software.

PAGE 42

35 Analysis of Pre-rRNA Processing by Pulse-Chase Labeling Approximately 25 OD600 units of cells in the mid-log phase (OD6oo~0.5) were collected, and resuspended in 5 ml of the synthetic minimal media. The cells were pulse labeled with 250 uCi [metfry/3 H]methionine or [ 3 H]uracil at 25C for 3 min. A large excess of unlabeled methionine (5 mM) or uracil (2 mM) was added. At the different chase time points, 1 ml of cells was collected and centrifuged for 10 seconds. Cell pellets were immediately frozen in dry ice and stored at -70C for further analysis. Total RNAs were extracted as described (Kohrer and Domdey, 1991). Briefly, cells were treated with 1% SDS and phenol at 65C for 5 min. After centrifugation, the supernatant was extracted with phenol ::CHCl3 and CHCI3 sequentially. The RNAs were precipitated by using 0.3 M NaOAc plus 2.5 volume of 100% ethanol. To increase the relative ratio of signal to noise, [ 3 H]uracil labeled RNAs were passed through oligo (dT)-cellulose (Boehringer Mannheim). The supernatant was transferred to a fresh tube and the RNA was ethanol precipitated, washed and dried. The RNAs were separated on 1.2% agarose-glyoxal gel or 12% polyacrylamide-urea gel (Ausubel et al., 1993). The gel was treated with En 3 Hance (DuPont NEN), dried and exposed to X-ray film. Northern Analysis of rRNAs Total RNAs were extracted, separated on 1.2% agarose-glyoxal gel, and transferred to Hybon nylon membrane (Amersham). The blot was probed with the [ 32 P] labeled oligonucleotides specific for the sequences in the internal transcribed spacer (ITS) 1 and 2, and in 1 8S and 25S rRNAs, and exposed to X-ray film. Before reprobing, the blot was submerged into boiling 0.5% SDS, cooled to room temperature, and washed with water. The oligonucleotides used as probes are listed in Table 4.

PAGE 43

36 Primer Extension Standard primer extension was performed as described below. Approximately 1 |ig of RNA template and 1 pmol of primer in the buffer (25 mM Tris-HCl, pH 8.3, 25 mM KC1, 5 mM MgCh, 5 mM DTT, 0.25 mM spermidine) were denatured at 70C for 10 min. and annealed at 42C for 20 minutes. Labeling was done for 1 minute at 42C in the presence of the followings: 0.5 mM each of dCTP, dGTP, and dTTP, 10 u€i of [a35 S]dATP, 5 units of avian myeloblastosis virus reverse transcriptase (Promega) and 20 units of RNase Block II (Stratagene). Extension was done for 10 minutes at 42C after adding dATP to 1 mM. The reaction was stopped by placing the reaction mixture on ice and adding 10 mM EDTA, and the nucleic acids were precipitated with ethanol prior to electrophoresis in 8 M urea-6% polyacrylamide gel. For concentration dependent pause (CDP) assay (Maden et al., 1995), the same procedure as the standard primer extension was performed, but with the concentration of dNTPs at 12.5 |iM each, and 20 uCi of [oc35 S]dATP during labeling. Extension was done with all four dNTPs at 4, 20, and 100 (iM. For partial alkaline hydrolysis of RNA (Sirum-Connolly and Mason, 1993), 2 fig RNAs were digested in 20 |il of 50 mM Na2CC>3, pH 9.0 at 90C for 6 minutes before standard primer extension. In vitro Methylation Analysis A 3.8-kb ApaUBamHl fragment containing T7 or T3 promoter followed by 27S rDNA sequence was linearized from pBluescript KS + or SK+ In vitro transcription was performed by using a commercially available kit (Promega). The reaction condition was set up according to the manufacturer's protocol. Both T3 and T7 RNA polymerase were successfully used. The reaction product was analyzed on a 0.8% agarose-glyoxal gel.

PAGE 44

37 Nop2p was obtained by immunoprecipitation (IP) with a monoclonal anti-Nop2p antibody (D68) (Wu, P., B. Hong, J. S. Brockenbrough, and J. P. Aris, unpublished results). When the IP pellet was analyzed by SDS-PAGE and stained with coomassie blue, a band corresponding to the position of Nop2p was seen (not shown). In vitro methylation reactions were performed as described (Segal and Eichler, 1991). The following components were contained in the reaction: 50 mM Tris-HCl (pH 8.0), 0.4 mM EDTA, 5 mM DTT, 0.4 mg/ml bovine serum albumin (BSA), 50 mM KC1, 2.0 U.M [ 3 H]-S-adenosyl-methionine (NEN), -50 uM 27S rRNA, 40 units of RNase Block (Stratagene), and IP-purified Nop2p. The mixture with the total volume of 100 |il was incubated at 30C for one to two hours. Considering the possible effects of secondary structure, denaturation of 27S rRNA by boiling before adding to the reaction was also tested. The reaction product was treated with phenol/chloroform. In the presence of 20 fxg of glycogen, the RNA was precipitated with 0.5 volume of 7.5 M ammonium acetate and 2.5 volume of 100% ethanol. The RNA was separated on a 0.8% agarose-glyoxal gel. The incorporated isotope was detected with fluorography. RNase Protection Assay RNase protection was done using a modification of a previously described method (Segal and Eichler, 1991). RNA was extracted from L4717 or YBH5 grown in SD for 8.5 hours, pulse labeled with [mef/ry/3 H]methionine for 3 minutes, and chased for 4 minutes, as described above. [ 3 H]labeled RNA from ~7 OD600 units of culture (-30 pmol RNA) plus 300 pmol of oligonucleotide probe were denatured in 40% formamide, 40 mM PIPESHC1, pH 6.4, 400 mM NaCl, 1 mM EDTA for 15 minutes at 85C. The oligonucleotides used for protection are as follows: oligo 6, oligo 7, and oligo 8 (Table 4). After cooling to 25C, single stranded RNA was digested with RNase A (20 p:g/ml) and RNase Tl (3 Mg/ml) freshly added to 300 mM NaCl, 10 mM Tris-HCl, pH 7.5, 5 mM EDTA.

PAGE 45

38 Digestions were inactivated with SDS and proteinase K, supplemented with 10 pg yeast tRNA, extracted with phenol-chloroform, ethanol precipitated, washed with 70% ethanol, and dried. Samples were electrophoresed on 12% polyacrylamide/7M urea gels. Gels were fixed and treated with En 3 Hance (DuPont). Following fluorography, sections of dried gels were excised, digested with 1 ml of Scintigest (Fisher Scientific) at 37C overnight in tightly capped glass vials. Samples were neutralized by the addition of 1 00 ixl of glacial acetic acid, mixed with Scintiverse E (Fisher Scientific), and subjected to scintillation counting for a sufficient length of time (0.5 8 hours per determination) to yield an error of 2% of the mean DPM value at a confidence level of 95%. Immunoprecipitation Analysis The buffers used in immunoprecipitation are listed as followings: IP buffer : 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, and 2 mM NaN 3 Lvsis buffer-C : 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, and 2 mM NaN 3 5 mM MgCl 2 0.1% NP-40 (Calbiochem), protease inhibitor cocktails in water (PIC-W) (1 mM benzamide, 0.5 pg/ml leupeptin, 1.5 pM bestatin in ddH20), protease inhibitor cocktails in dimethylsulfoxide (PIC-D) (0.5 mM phenylmethylsulfonyl Fluoride, 1 .5 pM pepstatin A, 1 pg/ml chymostatin in dimethylsulfoxide), 1 mM DTT, 5 mM vanadyl ribonucleoside complex. Pre-treatment buffer : 100 mM Tris-HCl (pH 9.0), 50 mM DTT, 5 mM EDTA. Lvsis buffer-N : 20% Ficoll, 20 mM KPj, pH 6.5, 1 mM MgCl 2 PIC-D and PIC-W. Cushion buffer : 30% Ficoll 400, 20 mM KPj, pH 6.5, 1 mM MgCl 2 PIC-W, and PIC-D. Washing buffer : 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM EDTA, and 2 mM NaN 3 5 mM MgCl 2 0.1% NP-40, PIC-W, and PIC-D. Monoclonal anti-Noplp (A66) and anti-Nop2p (D68), and polyclonal anti-Nop2p (APpAb3) antibodies were used. Binding of antibodies to protein-G sepharose (Pharmacia) was performed as followings: about 20 pi protein-G sepharose beads were

PAGE 46

39 washed with PBS buffer (2 mM KH 2 P0 4 8 mM Na 2 HP0 4 2.5 mM KC1, 140 mM NaCl, pH 7.2) plus 0. 1 % NP-40; after adding 5 ul of either A66 or D68, or 8 ul of APpAb3, the mixture was incubated at the room temperature (~25C) for 60 minutes; the beads were then washed three times with PBS buffer plus 0.1% NP-40. For each IP reaction, 5 OD600 units of L47 17 cells in the mid-log phase were used. An overnight culture of L47 1 7 cells was diluted into fresh YPD to an OD6oo~0. 1 After two doublings (OD600~0.4) at 30C, the cells were collected and washed with ddF^O. For protein labeling, the cells were incubated in 10 ml of SD medium (without methionine) at 30C for 1 hour. The cells were then collected and labeled in 1 ml of SD medium with -25 jnCi of [ 35 S]methionine at RT for 30 minutes. For RNA labeling, the cells were incubated in 10 ml of low-phosphorus-YPD (LPYPD) at 30C for 1 hour. Then the cells were labeled in 1 ml of LPYPD with -30 (iCi of 32 P-phosphate at RT for 30 minutes. For the whole cell lysate, labeled cells in 100 u,l of lysis buffer-C were broken by vortexing with RNase-free glass beads for 5 minutes at 4C. The supernatant was used for IPs. For nuclear preparation, labeled cells were treated with 100 (il of pre-treatment buffer. After washing with ddH20 and 1 M sorbitol, the cell wall was subjected to enzymatic digestion. For one OD600 of cells, 20 jig of Zymolyase 100T (ICN) and lul of glusulase (DuPont) were used. The digestion reaction was set up in 50 \i\ of 1 M sorbitol at RT for 30 minutes. The reaction was stopped by spinning for 30 seconds at RT at 7,000 rpm (4,000g), and the pellet was washed with cold 1 M sorbitol. Spheroplasts were homogenized in the lysis buffer-N using a Dounce homogenizer with 10 strokes at RT. The homogenate was centrifuged at 5,000 rpm (2,300g) in an SW50.1 rotor (Beckman) at 2C for 5 minutes. The supernatant was then loaded onto 1 ml of the cushion buffer. The nuclei in the supernatant were obtained by being centrifuged at 22,000 rpm (45,300g) in an SW50. 1 rotor at 2C for 60 minutes. The nuclear pellet was resuspended in 0.5 ml of lysis

PAGE 47

40 buffer-C and sonicated. After spinning at top speed in a microfuge at 4C for 5 minutes, the supernatant was used for IP. The supernatant from the whole cell lysate or from the nuclear preparation was mixed with the protein-G sepharose-antibody beads at 4C for 1 hour with rotation. The beads were washed five times with washing buffer. For protein analysis, the beads were resuspended in SDS-PAGE sample buffer (4% SDS, 20% glycerol, 0.1 M DTT, 83 mM Tris, 127 mM Tris-HCl, 0.001% bromophenol blue, pH 8.0), and boiled for 2 minutes. After a brief spin, the supernatant was loaded onto 10% SDS-PAGE, and analyzed by electrophoresis. For analysis of RNA-protein interactions, the RNAs were extracted from the beads as follows. Beads were resuspended in 150 u\l of IP buffer plus 1% SDS. In the presence of tRNA carrier, RNAs were extracted by 150 (i.1 of phenol: :CHCl3 at 37C for 15 minutes with vortexing for 1 minute every 5 minutes, and precipitated by ethanol. The extracted RNAs were separated on 7 M urea-6% polyacrylamide gel electrophoresis. The signals were detected by autoradiography. Screening for High Copy Suppressors A yeast library carried on 2 fim plasmid with URA3 marker was kindly given by Dr. Ruben Herriquez (Sandoz Pharmaceuticals AG). The library with an average of 10-kb inserts was prepared by Sau3A partial digestion and size fractionation. The library plasmids were transformed into E.coli DH5oc. Plasmid maxi-preparation was done as follows. An overnight culture of E.coli carrying plasmids was collected and treated with 1 .25 mg/ml of Lysozyme (Sigma) in 40 mis of GTE buffer (25 mM Tris, 10 mM EDTA, 50 mM glucose, pH 8.0) at RT for 10 minutes. Cells were disrupted with 80 mis of 0.2 N NaOH plus 1% SDS on ice for 15 minutes. After adding 60 mis of 3 M KOAc (pH 5.0), the mixture was placed on ice for 15 minutes, and centrifuged at 4C for 10 minutes at

PAGE 48

41 5,500 rpm (4,000g) in Sorvall GSA rotor. DNA in the supernatant was precipitated by adding 0.6 volumes of isopropanol, and centrifuged at 4C for 10 minutes at 4,000g. Pellet was dissolved in TE buffer, and mixed with 50 mis of 44% of CsCl plus 33% of ethidium bromide in quick-seal ployallomer tubes (Beckman). The mixture was centrifuged at 20C for 24 hours at 50,000 rpm (242,000g) in a VTi50 rotor. The bottom band containing plasmid DNA in CsCl gradients was collected and treated with ddF^Osaturated 1-butanol. DNA was obtained by ethanol precipitation. The library was then transformed into each of the six ts strains. In this study, it was found that almost no colony was formed if the transformants were incubated at 37C immediately after transformation. Therefore, the transformants were incubated at 25C for 24 hours before shifting to 37C. Since yeast genome size is about 14 Mb, it can be estimated that with this library, a minimum of 1,400 colonies for each ts strain is required for covering the whole yeast genome. The library plasmids were transformed into each of the six ts strains. The plasmids were recovered from the colonies formed at 37C, and amplified in E. coli DH5a. The suppression effect of the recovered plasmids was tested. Whether the recovered plasmids contained inserts was determined by restriction digestion with Pstl, Xbal, Nsil and EcoKV, which cut the plasmid backbone. Presence of URA3 marker in the recovered plasmids was determined by digestion with EcoKV /Nsil. Among about 100 plasmids recovered, only one contained a 3.7-kb insert. The SacVBgRl fragment containing 3.0-kb of the insert was then subcloned into pRS426 plasmid at the Sacl/BamRl sites, and its phenotypic effect was re-tested. Sequencing of the insert was done with the reverse primer, which was complementary to the poly linker 5' upstream to the insert. Another primer, JA36, complementary to the sequence of 25S rDNA, was also used for sequencing. Sequencing with -20 and -40 primers complementary to the polylinker 3' downstream to the insert was not successful. Whether the suppression was allele-specific was analyzed by testing the phenotypic effect of the insert in each of the six ts strains.

PAGE 49

42 Table 2. Yeast Strains Strain Genotypic description Source L4717 L4718 YBH3 YBH9 YBH12 YBH15 YBH16 YBH17 YBH18 YBH20 YBH21 YBH23 YBH26 YBH27 YBH28 YBH29 YBH30 YBH31 YBH32 YBH33 YBH34 YBH35 YBH36 MATa, ade2, canl-100, his3-ll,15 C. A. Style leu2-3,-112, trpl-1, ura3-l, GAL+ G. R. Fink MATa, ade2, canl-100, his3-l 1,-15, C. A. Style leu2-3,-\ 12, trpl-1, ura3-l, GAL+ G. R. Fink L47 1 7, nop2::LEU2, pJPA40 This study YPH501 nop2::LEU2 This study L4717, pJPA40 This study L47 1 7, nop2::LEU2, pBH60 This study L47 1 7, nop2::LEU2, pBH67 This study L47 1 7, nop2::LEU2, pBH72 This study YBH15, pJPA29 This study L47 1 7, nop2::LEU2, pBH55 This study L47 1 7, nop2::LEU2, pBH74 This study L47 17, nop2::LEU2, pBH77 This study YBH20, pJPA29 This study YBH16, pJPA29 This study YBH17,pJPA29 This study YBH21,pJPA29 This study YBH23, pJPA29 This study L47 1 7, nop2::LEU2, pBH49 This study L4717, nop2::LEU2, pBH56 This study L47 17, nop2::LEU2, pBH57 This study L4717, nop2::LEU2, pBH5S This study L4717, nop2::LEU2, pBH59 This study L47 17, nop2::LEU2, pBH6 1 This study

PAGE 50

43 Table 2. Continued Strain Genotypic description Source YBH37 L4717, nop2::LEU2, pBH62 This study YBH38 L4717, nop2::LEU2, pBH63 This study YBH39 L4717, nop2::LEU2, pBH64 This study YBH40 L4717, nop2::LEU2, pBH65 This study YBH41 L4717, nop2::LEU2, pBH66 This study YBH42 L4717, nop2::LEU2, pBH68 This study YBH43 L4717, nop2::LEU2, pBH69 This study YBH44 L4717, nop2::LEU2, pBH70 This study YBH45 L4717, nop2::LEU2, pBH71 This study YBH46 L4717, nop2::LEU2, pBH73 This study YBH47 L4717, nop2::LEU2, pBH75 This study YBH48 L4717, nop2::LEU2, pBH76 This study YBH49 L4717, nop2::LEU2, pBH78 This study YJA501 MATaJa, leu2A2, ura3-52, GAL2 J. Anderson YPH501 MATa/a, ade2-101a, his3A200, leu2Al, lys2-801a, trplA63, ura3-53, GAL+ R. S. Sikorski YSB-11 Diploid from cross of L4717 and L4718 J. S. Brockenbrough YSB12 YSB-11, nop2::HIS3 J. S. Brockenbrough STX466 MATa/a, ade2-lo, his3A200, lys2-801a, trplA ura3-52, gal Yeast genetic stock center 993313A MA 7a, his3A200, leu2-3,-U2, trplAl, ura3-52, GAL G. R. Fink 9933-20C MATa, his3A200, leu2-3,-J12, trplAl, G. R. Fink ura3-52, GAL 9933D Diploid from cross of 9933-20C and 9933-13A This study

PAGE 51

44 Table 3. Plasmids Plasmid Relevant functional DNA Comments pAH6 NOP2 HIS3 CEN6 pBH46 GAL10-NOP2 URA3 CEN6 pBH47 GAL10-NOP2 TRP1 CEN6 pBH49 NOP2 HIS3 CEN6 pBH55 nop2-3 HIS3 CEN6 pBH56 nop2-3a HIS3 CEN6 pBH57 nop2-3b HIS3 CEN6 pBH58 nop2-3c HIS3 CEN6 pBH59 nop2-3d HIS3 CEN6 pBH60 nop2-4 HIS3 CEN6 pBH61 nop2-4a HIS3 CEN6 pBH62 nop2-4b HIS3 CEN6 pBH63 nop2-4c HIS3 CEN6 pBH64 nop2-4d HIS3 CEN6 Sacl fragment containing NOP2 cloned at the same sites of pRS3 1 3 BamHl/Xhol fragment containing NOP2 from pJPA20 cloned in the same sites of pRD53 SacVXhol fragment containing GAL10-NOP2 from pBH46 cloned in the same sites in pRS314 Derived from pAH6 with deletion of Clal fragment from the upstream of NOP2 EcoKI fragment containing nop2-3 cloned at the same sites of pBH49 Mfel fragment from nop2-3 cloned at the same sites of pBH49 Mfel fragment from NOP2 cloned at the same sites of pBH55 Spel fragment from pBH55 cloned at the same sites of pBH49 Spel fragment from pBH49 cloned at the same sites of pBH55 E.coRl fragment containing nop2-4 cloned at the same sites of pBH49 Bsgl/Clal fragment from nop2-4 cloned at the same sites of pBH49 BsgVClal fragment from NOP2 cloned at the same sites of pBH60 Mfel fragment from NOP2 cloned at the same sites of pBH62 Mfel fragment from nop2-4 cloned at the same sites of pBH49

PAGE 52

45 Table 3. Continued Plasmid Relevant functional DNA Comments pBH65 nop2-4g HIS3 CEN6 pBH66 pBH67 pBH68 pBH69 pBH70 pBH71 pBH72 pBH73 pBH74 pBH75 pBH76 nop2-4h HIS3 CEN6 nop2-5 HIS3 CEN6 nop2-5a HIS3 CEN6 nop2-5b HIS3 CEN6 nop2-5c HIS3 CEN6 nop2-5d HIS3 CEN6 nop2-6 HIS3 CEN6 nop2-6a HIS3 CEN6 nop2-9 HIS3 CEN6 nop2-9a HIS3 CEN6 no P 2-9b HIS3 CEN6 Derived from pBH62. Mfel fragment deleted, and the gapped plasmid ligated at the same site. ClaVMfel fragment from pBH49 cloned at the same sites. Mfel fragment from nop2-4 then cloned at the same site. Derived from pBH49. Mfel fragment deleted, and the gapped plasmid ligated. ClaVMfel fragment from pBH63 cloned at the same sites. Mfel fragment from nop2-4 then cloned at the same site. E.coRl fragment containing nop2-5 cloned at the same sites of pBH49 Mfel fragment from nop2-5 cloned at the same sites of pBH49 Mfel fragment from NOP2 cloned at the same sites of pBH67 Spel fragment from pBH67 cloned at the same sites of pBH49 Spel fragment from pBH49 cloned at the same sites of pBH67 E.coRl fragment containing nop2-6 cloned at the same sites of pBH49 Bsgl/Mfel fragment from nop2-6 (one of two Mfel sites lost from the mutation) cloned at the same sites of pBH49 E.coRl fragment containing nop2-9 cloned at the same sites of pBH49 Mfel fragment from nop2-9 cloned at the same sites of pBH49 Mfel fragment from NOP2 cloned at the same sites of pBH74

PAGE 53

46 Table 3. Continued Plasmid Relevant functional DNA Comments pBH77 nop2-W HIS3 CEN6 pBH78 nop2-10a HIS3 CEN6 pJJ282 LEU2 pJPA20 NOP2 pJPA29 NOP2 TRP1 CEN6 EcoRl fragment containing nop2-10 cloned at the same sites of pBH49 BsgVMfel fragment from nop2-10 (one of two Mfel sites lost from the mutation) cloned at the same sites of pBH49 Jones, J. S. and L. Prakash ZscoRI fragment containing NOP2 cloned into pBluescript SK + XhollBgRl containing NOP2 cloned at XhoVBamHl of pRS3 14 pJPA40 NOP2 URA3 CEN6 HpaVKpnl fragment containing NOP2 plus 715 bp of 5' flanking sequence and 456 bp of 3' flanking sequence from pJPA20 cloned into pRS316 pRD53 GAL1, 10 R. J. Deshaies pRS426 2um URA3 T. Christianson

PAGE 54

47 Table 4. Oligonucleotides Name Sequence JA36 Oligol (18S) 01igo2(ITSl-U) 01igo3(ITSl-D) 01igo4 (ITS2-D) 01igo5 (25S) 01igo6 5' 01igo7 5' 01igo8 5' 01igo9 (A) OligolO (B) Oligol 1(C) Oligol 2(D) Oligol 3(E) Oligol 4 (JA22) Oligol 5 (JA30) 01igol6(JA31) P3 P4 P5 P6 P7 P8 P9 5 -GGTGGGTTAGTCGATCC-3 5 AGCC ATTCGCAGTTTC ACTG-3 5'-GCACAGAAATCTCTCACCGT-3' 5'-TCCAGTTACGAAAATTCTTG-3' 5 -GCCTAGACGCTCTCTTCTTA-3 5 -TACTAAGGC AATCCGGTTGG-3 CGCATAGACGTTAGACTCCTTGGTCCGTGTTTCAAGACGG-3' CCCTATTAGTGGGTGAACAATCCAACGCTTACCG-3' CGGTCTAAACCCAGCTCATGTTCCCTATTAGTGGG-3' 5'-CCTTAGAGCCAATCCTTATC-3' 5 -CTGTTGACGTGG AACCTTTC-3 5 -CTGATGAGCGTGTATTCCGG-3 5 -C AGG AACC AGCTACTAGATG-3 5'-CACTTTCATTACGCGTATGG-3' 5'-CATCAGTAGGGTAAAACTAAC-3' 5 -TGTTCTACTGGAGATTTCTG-3 5 -TAGGG AC AGTGGGA ATCTCG-3 5 -GCGGAGA AC ATCC AAC-3 5' -AGATCGGTCCATCTTCG-3' 5 -GGTTGC ATGTTCTCCGC-3 5'-CCTTGAAGACCAGAAGG-3' 5 -CG AAGATGGACCGATCT-3 5 -GTACCGGA AC ATGGGGC-3 5'-TGAAGCAGGATTGGTGG-3'

PAGE 55

48 Table 4. (continued) Name Sequence P10 P12 P16 P19A P21 P23 P24 P25 P26 P27 P28 P29 Reverse -20 -40 5 -TC ATCC ACC AATCCTGC-3 5'-CTATGCTAACATGATGC-3' 5'-TTCCTTCTACCGGTACC-3' 5 -GCCGG ACGAT AACGTGC-3 5'-CTAAGTCTCTCCTTCTG-3' 5 AAGAACAAGC AAGCCGC-3 5 -GTACCGAG AAGGACTTC-3 5'-GAATTCCCCTTGTGTTG-3' 5'-AGCGAAAACACAACCTG-3' 5 -GAATCAATGGCGGATAG-3' 5 -TGCTCTAG ATCCTCATG-3 5'-ACCGAATGTCAAGTTAG-3' 5'-AACAGCTATGACCATG-3' 5'-GTAAAACGACGGCCAGT-3' 5 -GTTTTCCC AGTC ACGG AC-3

PAGE 56

49 Table 5. Cell Viability after Nop2p Depletion Time of Depletion (Hour) Colony Number (Depleted) (Non-depleted) 157 155 1 152 150 2 149 154 4 151 155 8 147 156 10 144 159 23 145 162 25 140 155 Survivability (%) 101 101 97 97 94 91 90 90

PAGE 57

RESULTS Depletion of Nop2p Repression of NOP2 Impaired Cell Growth When expression of a gene is repressed, the protein encoded by the gene will be depleted because of protein turnover and dilution by cell division. In this way, a deficit in the protein function will be revealed. To repress the activity of the GAL promoter and deplete Nop2p, YBH5 (GAL-NOP2) cells were shifted from YPGal to YPD medium. During the first 8 hours after the medium-shift, YBH5 cells grew at a similar rate in both media. However, after 8 hours' incubation, the cell growth in YPD medium was progressively impaired, as compared with that in YPGal medium (Figure 6A). The doubling time in YPD medium increased about fivefold from 2.5 hours at the medium-shift to 12.5 hours after the medium-shift for 28 hours. To confirm that Nop2p in the YBH5 cells grown in YPD medium was indeed depleted, Western blotting with an antibody to Nop2p was performed. At the 2 hour time point after medium-shift, the Nop2p level was significantly reduced. After 4 hours' depletion, Nop2p was not detectable (Figure 6B). The cytoplasmic poly(A) binding protein Pablp level was not affected during the incubation in YPD, suggesting that Nop2p was specifically depleted (Figure 6B). Since a significant depletion of Nop2p occurred before the cell growth slowed down, the cell growth was dependent upon the function of Nop2p. 50

PAGE 58

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PAGE 59

52 s r= c CD o 1 w w in CO C\J 1 — T T -o tXjjsuea iieo

PAGE 60

53 Depletion of Nop2p Caused a Reduction in 25S rRNA and 60S Ribosomal Subunits The nucleolar localization of Nop2p (de Beus et al., 1994) was a clue that it might be related to nucleolar function. The essentiality of this protein to cell growth and viability might be due to its involvement in the ribosome synthesis, one of the main functions in the nucleolus (Hadjiolov, 1985). To investigate this possibility, the relative levels of 1 8S and 25S rRNAs in Nop2pdepleted cells were measured. Prior to Nop2p depletion (at time point), the ratio of 25S and 18S rRNAs was close to 1:1 (Figure 6C). However, after 28 hours' depletion of Nop2p, 25S rRNA level was significantly reduced and the ratio of 25S to 1 8S rRNAs was much less than 1 : 1 (Figure 6C). Over 90% of YBH5 cells grown in YPD for 28 hours were still viable (Table 5). Therefore, the observed reduction of 25S rRNA was not caused by cell death. Since 25S rRNA is a structural component of 60S ribosomal subunit, these results suggest that Nop2p is involved in synthesis of the 60S ribosomal subunit. To confirm the role of Nop2p in ribosome synthesis, the effect of Nop2p depletion on the levels of ribosomal subunits and polyribosome was analyzed. A typical, normal polysome profile was shown in the control L4717 cells (Figure 7A and B). In contrast, depletion of Nop2p for 4.5 hours caused a reduction in the level of 60S subunit relative to that of 40S subunit. The peak of 80S monomer, which consists of the 40S and 60S subunit associated with an mRNA, was accordingly lowered. Three small extra peaks appeared immediately following the peaks of 80S monomer and the first two polysome (Figure 7C). These extra peaks possibly represent halfmer polysomes, which contain the mRNA-associated polysomes plus a preinitiation complex consisting of 40S subunit with the initiation factors (Helser et al., 198 1 ; Moritz et al., 1990). A similar abnormal polysome profile was observed when synthesis of 60S ribosomal subunit was aborted by depletion of large subunit proteins LI or L16 (Moritz et al., 1990; Deshmukh et al., 1993).

PAGE 61

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PAGE 62

55 o in Q Q_ >I CD > E CQ LUU ci ci LUU SZ V O in Q 0> c LO X CD > & < 2 in d O 5 ci LUU frSZy LUU frSSy

PAGE 63

56 Since the growth rate of the Nop2p-depleted cells was similar to that of the non-depleted cells during the first 8 hours of growth in YPD, the aberrant polysome profile was not due to a secondary effect of slow growth. After the medium-shift to YPD for 12.5 hours, the level of the 60S subunit was significantly reduced while the 40S subunit accumulated (Figure 7D). The reduction of 80S monomer was more significant and the halfmer peaks were more obvious, suggesting that the shortage of the 60S subunit was much more severe after a longer period of depletion (Figure 7D). These observations indicate that Nop2p is required for synthesis of the 60S ribosomal subunit. Nop2p is Required for Processing of 27SB Intermediate into 25S and 5.8S rRNAs The fact that Nop2p depletion caused a reduction in 25S but not 18S rRNA suggests that the protein might be involved in processing of pre-rRNA. To investigate this possibility, YBH5 (GAL-NOP2) cells grown in YPD for 8.5 hours were labeled with [methyl 3 H] methionine, and chased with an excess of methionine. Since yeast rRNAs are methylated at a large number of sites soon after transcription (Brand et al., 1977), following the labeled methyl groups on the pre-rRNA to the mature rRNAs can reveal the intermediate steps of pre-rRNA processing. In the control L4717 cells, 35S pre-rRNA was almost immediately processed into 27S and 20S pre-rRNAs (Figure 8B). At 4 minutes of chase, most 27S and 20S pre-rRNAs were processed into 25S and 18S rRNAs, respectively. At 8 minutes of chase, almost all of these pre-rRNAs were converted to mature rRNAs (Figure 8B). However, in the Nop2p-depleted cells, 35S and 32S prerRNAs significantly accumulated at 2 minutes of chase. Even at 8 minutes of chase, a large amount of 27S pre-rRNA still accumulated with a significant reduction in the formation of 25S rRNA, which is consistent with the finding that the steady-state level of 25S rRNA was reduced after 28 hours of Nop2p depletion. Nevertheless, the processing of 20S to

PAGE 64

Figure 8. Depletion of Nop2p results in a defect in pre-rRNA processing. YBH5(GAL-NOP2) (A) and L4717 (NOP2) (B) were shifted from SGal to SD for 8.5 hours, pulse-labeled with [ 3 H-metfry/]methionine, and chased with excess methionine for 0, 1, 2, 4 and 8 minutes. Total RNA was extracted, separated on an agarose-glyoxal gel, and detected by fluorography. Approximately one-third of the L4717 8-min sample was loaded on the gel.

PAGE 65

58 A B YBH5 (GAL-N0P2) L4717 (N0P2) 12 4 8min. 12 4 8min. 32S^ 27S25S^ -, fi-SS 20S^ 18S^ •-•=k

PAGE 66

59 18S rRNA in Nop2p depleted cells was similar to that in the control cells (Figure 8A). These results suggest that Nop2p is involved in the processing pathway leading to 25S rRNA. In this pulse-chase assay, the processing of pre-rRNA was evaluated by following incorporation of label into methyl groups, which had been transferred onto pre-rRNA. Therefore, the possibility had to be excluded that the aberrant processing might be actually a change in the methylation pattern. To do this, [ 3 H]uracil was used to label the backbone of pre-rRNA for pulse-chase analysis. In the control L4717, 35S and 32S precursors were quickly processed (Figure 9B). Chasing of the 27S pre-rRNA to 25S rRNA in the control cells took a longer time than in [methyl 3 H] labeling. This is possibly because the cell can re-use the labeled uracil resulting from the turnover of RNAs within the cell for a quite long time (Warner, 1991). In YBH5 depleted of Nop2p for 8.5 hours, a significant amount of 35S and 32S precursors were not processed even at 8 minutes of chase (Figure 9A). Compared with the control, a significant delay in the processing of 27S pre-rRNA to 25S rRNA was observed at 8, 16, and 32 minutes of chase, with the formation of 25S rRNA accordingly reduced (Figure 9A). Similar to what was observed in [methyl 3 H] pulsechase labeling, the processing of 20S to 18S rRNA was not significantly affected by Nop2p depletion. These results confirm that Nop2p is required for processing of prerRNA. To locate the specific processing step in which Nop2p might be involved, the steady-state levels of pre-rRNAs and rRNAs were estimated by Northern analysis. With probes specific for regions in the ITS 1 and ITS2, and in the 1 8S and 25S rRNAs (Figure 10), the various processing intermediates can be recognized. Consistent with the results from the pulse-chase analysis, Nop2p depletion for 4.5 and 8 hours caused a significant accumulation of the steady-state levels of 35S and 32S precursors, as compared with the non-depletion cells (at time point) and the controls YBH3 and L4717 (Figure 1 1 A, B, and C). With the probe (oligonucleotide 3) complementary to the 27SA but not 27SB

PAGE 67

Figure 9. Pulse-chase labeling analysis of pre-rRNA processing by using [ 3 H]uracil. YBH5 (GAL-N0P2) (A) and L4717 (NOP2) (B) were grown in SGal, shifted to SD for 8.5 hours, pulse-labeled with [ 3 H]uracil, and chased with excess uracil for 0, 2, 8, 16, and 32 minutes. The samples were treated with oligo (dT)-cellulose before agarose-glyoxal gel electrophoresis.

PAGE 68

61 B YBH5 (GAL-N0P2) 2 8 16 32 L4717 (N0P2) 2 8 16 32 35S"~ 32S*27S^ 25S*~ 20S18S"~ • 0

PAGE 69

v: IT) ri CO •— <*. o O 13 B B O a3 "el o 3 -a P 3 o cx 4—' 1) '3 = 5 u c3 /"™n. .c O 00 4— g c o c IS) T3 C "*- a 3 < O a o T3 § Z 4-* i— "o 51 C 00 C3 c "Eg <£ o '4—* "3 'S <4— O" _W) 02 4-J 2 'e/3 c3 oo E a +— cn S t> T3 9 '_ 4- ;/; C 3 Wj zj i> _c w-> — ex 'c/3 t/3 C — < CO > -o u „ c rifl fN a c3 < 00 r4—* 4-t n c/3 C o O .2? J 00 *4— o "o "5 60 8 oo" ex 4-* O la CI ex 3 — < •— o o Z < Z z e o 4- J5 C4 as u a. -C 5 — 1 3 E Z u ,0 ex — 3 3 4— X) 8 a no OJ c/3 >> u 4—* 3 4-> p 3 1/3 5 O i/i
PAGE 70

63 CM a o CO hLU 00 < CO CM eg CDCM CO ICO O. CM o — CO m. Ihs. Ico CO O CO O JD ol o |co l CO Jg O CD CO < CO CD .E CO CO CO CO > a) co o 02 o O a! CO A CO < CD CO CO O c o X LU II CM o CD CO CO > CO O O o CD O) CO > CO O LI t\ 0) Ml n 1 CO i CO A C/J CD CM CO 1^ o U 00 C U) o X LU CO hLU LO CO 00 Io <. CO LO CO CD CO CO > co _CD O en o CM co 00

PAGE 71

E c cn p p 4— 60 '$ o r~13 J2 y — i X O* B U < CQ ;_J ? u 13 — £ o E u E O •— 0. H a! T3 c 60 1 >1 h— 1 < Q c cn Z a. r3 4-t U !D o 3 2 fc o 9 g 'X v. c 3 E 1 E a o -a 9 — 4- \S £ c t3 "u o z E &0 s c/5 "3 X -> 5) o ex 1— 5< a — o c<3 -4-* u o CN CO CN C '5 H w-i — "3 CI, -3 C C3 1o c u 13 <-> jr B C 1 'a? o o X) *> i 00 13 B 13 — o 3 o ^> CS c3 B on o n O 00 93 >/") D, W) — S U v. "3 =_ d H > — !> C *-< N — 13 .— 73 o u < _0J 3 c C5 z 3 a ^ o 9 C o C o X5 OB CL, o b. 9 OX) X CQ Q Cu £ P _B

PAGE 72

65 L\LX>-\ £H9A !C C *\" C o i =s =1 t I m £H9A !fl O if £H9A 0CO !£ O §2 < C o St 11 coco
PAGE 73

66 pre-rRNA, the accumulation of 27SA pre-rRNA was observed in the Nop2p-depleted cells (Figure 1 IB). Nop2p depletion also caused a significant accumulation of 27SB pre-rRNA (Figure 1 1 A). The pathway leading 32S precursor to 23S and 27SB intermediates is a minor alternative one in wild type cells (Russell and Tollervey, 1992). Previous studies demonstrated that depletion of Noplp (Tollervey et al., 1991), or Garlp (Girard et al., 1992), or snoRNA U3 (Tollervey, 1987) caused the accumulation of 23S. Depletion of Nop2p had no significant effect on the level of this intermediate (Figure 1 1C). The level of 20S pre-rRNA, a precursor for 18S rRNA, was similar among Nop2p-depleted cells and the control cells (Figure 1 1C). The sample loading was adjusted according to the levels of 25S and 18S rRNAs (Figure 1 ID). The above results indicate that Nop2p functions at the step of processing of 27SB to 25S rRNA (Figure 10). If this is true, then the formation of 5.8S rRNA should also be affected by Nop2p depletion, since 27SB is the common precursor for 25S and 5.8S rRNAs (Figure 10). To test this assumption, the processing of 5.8S rRNA was evaluated by pulse-chase analysis. At 10 and 30 minutes of chase, the formation of 5.8S rRNA was reduced in YBH5 cells depleted of Nop2p for 8.5 hours, as compared with that in the control L4717 (Figure 12A). Approximately equivalent loading was estimated by staining the gel with ethidium bromide (Figure 12B). Since 5S rRNA is transcribed independently by RNA polymerase III, its level should not be affected by the defect in the processing of the large rDNA transcript (Raue and Planta, 1991). A similar rate of the formation of 5S rRNA was observed in YBH5 and the control L4717 cells, suggesting that Nop2p depletion specifically affected the formation of 5.8S rRNA (Figure 12A). In summary, the results from the pulse-chase analysis and Northern blotting indicate that Nop2p is required for processing of pre-rRNA, specifically at the step of conversion of 27SB pre-rRNA to 25S and 5.8S rRNAs. However, the formation of 18S rRNA from 20S precursor is relatively independent of Nop2p function.

PAGE 74

2 w ro P o 3 £ 1 g ft o 1 1 5 71 -a u i u 35 on P 9 O £ < S 3 8 n *- ^^ -rj O s § 3 Z £ VI S .3 a 3 IB x: 3 51 o e -= — u-i u oo I s -8 1/3 VI 3 OB cu r< a o Z c— O c a Q Q 00 a u * E "3 o & u-Q E 3 cd T3 c j= o aj .c a rr* 1 \ CD 95 T3 J3 "3 a C — X cm So P r ^ C 'a c/1 t/j 53 O i! J OJ I) E Bi CD -C a 5 o Ea bO U -0 13 bd c 1 u J3 a E o '*— CO US u TD C ea o H

PAGE 75

68

PAGE 76

69 Mutational Analysis of Nop2p Another approach to investigate the function of a protein is to make mutations in the protein. A protein containing point mutations may assume a non-functional conformation at a restrictive condition. If the gene is essential, the mutations will cause a lethal phenotype at a restrictive condition, but not at the permissive condition. At a certain restrictive condition, global changes in cellular processes (cytoskeleton, protein synthesis, replication, transport, etc.) may occur, also causing cell death. However, this possibility can be excluded if the lethal phenotype can be rescued by the gene encoding this protein. One advantage of this approach is that all of the mutated protein generated at a restrictive condition will be non-functional, while it takes a much longer period of time to deplete a protein. Therefore, a deficit in the function will be caused more likely by the mutated protein than by secondary effects. Another advantage is that different alleles of a gene may cause different phenotypes, which helps to reveal multiple functions of the gene product (Huffaker et al., 1987). Isolation of the Six Temperature-Sensitive nop2 Alleles In this study, random point mutations in NOP2 were generated in vitro by using a low fidelity PCR method (Muhlrad et al., 1992). The phenotypes of the cells containing only the mutated nop2 alleles were analyzed under three restrictive conditions. One is temperature, which has been widely used to isolate conditional mutants (Huffaker et al., 1987; Tollervey et al., 1993). At low (14C) or high (37C) temperatures, it may be more demanding for a protein to maintain its functional conformation. Mutation at critical site(s) of the protein can disrupt the functional conformation, causing a mutant phenotype at either high (Ts) or low (Cs) temperatures. Recently, formamide and ethanol were found to be very useful for isolation of conditional mutants (Aguilera and Benitez, 1986; Aguilera,

PAGE 77

70 1994). The chemical groups (C=0, OH, NH) present in formamide and ethanol may compete for noncovalent bonds with the amino acid residues of the protein. Mutation in a protein may weaken the non-covalent bonds formed among the amino acid residues because the amino acid substitution may alter charges or hydrophobicity at the local region. In the presence of formamide or ethanol, the functional conformation of a mutant protein may be disrupted, resulting in either formamide-sensitive (Fs) or ethanol-sensitive (Es) phenotype. Two mutagenic PCR conditions were tested to generate mutations in the NOP2. One includes the addition of 0.15 mM of MnCl2 and a bias in the nucleotide pools. Another includes the bias in the nucleotide pools only. The PCR products and the gapped plasmid pBH49 were cotransformed into YBH3 cells. After plasmid shuffling (see MATERIALS AND METHODS), the effects of the mutations were tested. With the first condition, only very few colonies (an average of 40-50/plate) formed at 25C (not shown), suggesting that most of the nop2 alleles were non-functional even under the permissive condition. An estimate of 10,000 colonies carrying the PCR products from the first condition was screened for Ts and Cs phenotypes. However, no conditionally lethal strain was found. With the second condition, an average of 150-200 colonies/plate formed at 25C. In order to increase the chance of isolating conditionally lethal strains, two additional restrictive conditions, 3% formamide and 6% ethanol, were used. A total of 22,000 colonies carrying the PCR products from the second condition was screened. Some Ts, Cs, and Fs/Ts candidates were isolated, but no Es or Fs only candidate was found (Table 6). To exclude the possibility that the phenotypes might be caused by mutation in other genes on the chromosomes, the plasmids in these candidates were recovered, and their phenotypic effects were tested again in the same genetic background (Table 6). Among the twenty-six Ts candidates, the majority (73%) were caused by the mutation on the chromosomes. No Cs phenotype was found to be plasmid-borne. The plasmid recovered from the Fs/Ts candidate still exhibited the phenotypes (Table 6). To make sure that it was

PAGE 78

71 Table 6. Screening for Conditionally Lethal nop2 Alleles Phenotype Candidates Plasmid-borne phenotype ORF-borne phenotype Ts 26 Cs 2 Fs/Ts 1 Es 7 1 5 1 Ts: temperature-sensitive. Cs: cold-sensitive. Fs: formamide-sensitive. Es: ethanol-sensitive. ORF: open reading frame.

PAGE 79

72 the mutation in the protein that caused the phenotypes, the open reading frame (ORF) of the alleles was subcloned and its phenotypic effect was tested again. A total of six conditionally lethal alleles were isolated. All six alleles conferred the cells with the temperature sensitive phenotype, while they still supported cell growth to some degrees at the permissive temperature (25C) or at 30C (Figure 13). In liquid medium, 8 hours after the temperature shift, the growth curve of the six Ts strains already reached a plateau, while the wild type L4717 still grew in log-phase (Figure 14). Interestingly, nop2-10 strain expressed not only Ts, but also Fs at 25C (Figure 15, nop2-10), which suggests that the mutation at an additional site(s) of this allele caused this additional phenotype that was not expressed in the other Ts strains. The conditionally lethal phenotype of the six Ts strains was rescued by another plasmid carrying the wild type NOP2 gene (Figure 15 and not shown). This confirms that the phenotype was caused by mutation in NOP2. To exclude the possibility that the phenotype might be due to rapid protein degradation caused by the mutations, immunoblotting analysis of the mutated versions of the protein was performed. After 6 hours at 37C all of the six mutated versions of the protein were present, though the amounts were relatively lower than the two controls (L4717 and YBH3) (Figure 16). This result suggests that the mutant proteins were relatively stable. Compared with the two controls (L4717 and YBH3), there was no change in protein mobility of nop2-3 and nop2-4. However, nop2-5 protein moved faster than the other mutants and the controls (Figure 16). Sequencing of this allele revealed that there was an amber mutation near the 3' end of the ORF, causing a truncation of 14 amino acids at the C terminus (see Figure 19). There was also a change in the protein mobility of nop2-6, nop2-9 and nop2-10 (Figure 16), which may be due to changes in charge or modification of the mutant proteins. The above results indicate that the phenotypes of the six nop2 alleles isolated are due to the mutations in the ORF of the gene and that the amino acid substitutions in the mutant proteins result in disruption of the functional conformation of the protein at the

PAGE 80

Figure 13. The effect of temperature on the growth of the six nop2 temperaturesensitive (ts) strains. Serial five-fold dilutions of the cells of L4717, YBH3, and the six nop2 ts strains (nop2-3, nop2-4, nop2-5, nop2-6, nop2-9 and nop2-10) were grown on YPD plates at 25C, 30C or 37C.

PAGE 81

74 2 5C 30C 37C L4717 #• H • • ft + •#* • YBH3 # # • t> • •• t • ••4 nop2-3 # # t< ; • • ** nop2-4 0| ,. • <* nop2-5 01 | f • • :• < nop2-6 ||K • • nop2-9 # # £ '•' • • K• nop2-10 • N y

PAGE 82

Figure 14. Growth curves of the six nop2 ts strains. The cells of L47 1 7 and the six nop2 strains (nop2-3, nop2-4, nop2-5, nop2-6, nop2-9 and nop2-10) were grown in YPD at 25C and shifted to 37C (at time). At the indicated times, the cell densities were measured (A60o)-

PAGE 83

76 10003 100j o — -oA---ffl---nop2-3 nop2-4 nop2-5 nop2-6 nop2-9 nop2-10 L4717 0.01 Hours at 37C

PAGE 84

Figure 15. Mutations in NOP2 caused temperature sensitive and formamide sensitive phenotypes. The cells of nop2-9 and nop2-10, and the corresponding "rescue" strains (carrying pJPA29 plasmid) were streaked on YPD plates with or without 3% formamide, and incubated either at the restrictive (37C) or the permissive (25C) temperature as indicated. The growth of wild type L4717, and YBH3 (carrying plasmidborne NOP2) are shown as controls.

PAGE 85

78 nop2-9 / / BM3 37C 25C nop2-10 25C FA ft 25C + FA 37C FA

PAGE 86

Figure 16. Mutant nop2 proteins are relatively stable. The cells of L4717, YBH3 and the six nop2 ts strains (nop2-3, nop2-4, nop2-5, nop2-6, nop2-9 and nop2-10) were grown in liquid YPD at 37C for 6 hours. Total cellular proteins were extracted and analyzed by Western blotting with a polyclonal antibody to Nop2p. The position of Nop2p is indicated. Non-specific bands are labeled with stars.

PAGE 87

80 Tfr CD nop2 alleles > 3 4 5 6 9 10 Nop2p-

PAGE 88

81 restrictive conditions. Since nop2-10 allele caused Fs, in addition to Ts phenotype, several structural domains may exist in the protein. Mutation in some domains may result in Ts phenotype only, as was shown in the other five nop2 alleles, while mutation in other domains may cause Fs or both Ts and Fs phenotypes. The Ts or Ts/Fs phenotypes are most possibly caused by the direct effects of mutation in NOP2 gene, though it is also possible that mutation in NOP2 may make the cells extremely "sick" and that a second "hit" with heat or formamide may actually cause cell death. Characterization of the Six nop2 Alleles One advantage of using a mutational analysis approach is that it can reveal multiple functional domains in a protein. Since each of the six nop2 alleles contains multiple point mutations, analysis of the effects of these different mutations would help understand more about the protein's function. Each allele was analyzed for its effect on the synthesis of ribosomes. The six Ts strains and the control YBH3 strain (carrying plasmid-borne NOP2) were incubated at 37C for two hours, and the levels of ribosomal subunits and polysomes were measured. In the control YBH3, incubation at 37C did not cause any significant change in the polysome profile (not shown). In contrast, even at the permissive temperature (25C), all of the Ts strains, except nop2-3, already exhibited a significant change in the polysome profile, including a reduction in the peaks of the 60S subunit and the 80S monosome, a relative accumulation of 40S subunit, and appearance of halfmer polysomes (Figure 17). At 37C for 2 hours, a more severely abnormal polysome profile was seen in all of the six Ts strains (Figure 17). However, the degree of the severity in the defect appeared to vary among the six Ts strains, with nop2-10 strain affected most. When YBH3 was incubated in the medium containing 3% formamide at 25C for two hours, the peaks of both 40S and 60S subunits were proportionally reduced. However, there was no change in the peaks of 80S

PAGE 89

1 — o u in Q £• CN £ c 0-> M f, ua ^ 1 CM | 3 4-1 u. U a, E c o c M c/5 E o o j-j 5 _>^ '3 •— u m o o m X T3 \r. CN CO a fed U P n 1 > j/3 E c 3 o X — O o £ CJ U CN 'C -C Pi — o H 5 a c o • — • U u E c IT) 18 3 rcn 4— 1 XI eo -*— > '£ 3 1 — c3 3 H u -D mm ^ > 3 '*— J-. E u 1 E c 'C *— > cd s J-. E o c o :/: ,2 c O O y3 y '-jjg is M # "C O v Cm M Q bo P O "S u > H *— c M C -3 3 3 o o 5 Q 6o H a. **^ c c C V3 < c O i W E < T3 N c u 3 > "C a eg 3 e cd
PAGE 90

83 i o m w?V W2V W2y E o C o CO wzv E a o wzy O K CO o CM Q. O c o fl o in frS2y O o LO CM O I CM d o c E i o m wsy W2y wgy

PAGE 91

84 monomer and polysomes, and no halfmer polysome was seen (Figure 17). When incubated in the medium containing 3% FA at 25C, only nop2-10 strain showed a similar abnormality that was seen when incubated at 37C (Figure 17 and not shown). The results confirm that Nop2p is required for the synthesis of 60S ribosomal subunit. Since there are different degrees of severity in the defect caused by these six alleles, and only nop2-10 strain showed the formamide-sensitive phenotype, it is possible that several regions within Nop2p may be required for the functional conformation. The effects of the six Ts alleles on pre-rRNA processing were evaluated by pulsechase labeling analysis. After 2 hours at 37C, all of the six Ts strains (nop2-W not shown) exhibited a significant accumulation of 35S and 32S precursors. At the same time, the formation of 25S rRNA was significantly delayed and reduced (Figure 18). In the control YBH3 1 (carrying plasmid-borne NOP2) grown under the same conditions, the processing of pre-rRNA was rapidly completed (Figure 18). There was no accumulation of 20S pre-rRNA and the formation of 18S rRNA was not significantly affected in the six Ts strains at 37C (Figure 18). When grown in 3% formamide medium at 25C, nop2-10 strain also exhibited a similar defect in the processing that was observed at 37C (Figure 18). However, when grown in the medium without 3% formamide at 25C, the processing in nop2-10 appeared to be similar to that in the control YBH31 (Figure 18). These results are consistent with those from Nop2p depletion analysis. However, the 27S processing intermediate rapidly disappeared in nop2-4 and nop2-5 strains, though the formation of 25S rRNA in these two strains was also reduced (Figure 18). In all of the other Ts strains, accumulation of this intermediate was significant, even at 8 minutes of chase (Figure 18). Since Nop2p was involved in the methylation of only one site on 25S rRNA sequence (see below), It is most possible that the 27S intermediate in nop2-4 and nop2-5 strains was rapidly degraded. These results confirm that Nop2p is required for the processing pathway leading to 25S rRNA. The fact that 27S processing intermediate did not accumulate in nop2-4 and nop2-5 strains suggests that the region(s) mutated in nop2-4 and nop2-5 alleles

PAGE 92

nrj P 3 O -3 H u 3 3 E & on o r^i E (3 ad e 1 CN O CQ 3 -a ~ • — 6£ u c 3 o C • •a qj o oc o SB "o u — o c O 0) x: a 0 00 g a 3 -2 c o E o IE 4— 13 bQ 3 2 1 E 15 X o QC SI 1 si 1 "5b u J-. 3 i i rs 1 Qg 5. ft. ^ c ^ bo s c C 3

PAGE 93

S6 CM CL O c CM a. o CO i ED > II I II It AA WW iocm mm i: I I i I I A A w w r in CM CM I I 1 I ) A A w w o co CM tO o in CM O CM a o u + o CM Q. O CM Q. O 1 1 1 1 • 1 1 1 1 1 1 It 1 J 1 I J $ 1 II 1 m 1 i II J fl II • 1 m II 1 t i: i AA WW IOCM CD CO A A w w CM CM A A w w O 00 CM i-

PAGE 94

87 was required for the stability of 27S intermediate. It is possible that certain region(s) in Nop2p may be involved in the association of other proteins or snoRNPs with 27S intermediate and that disruption of such association may subject 27S intermediate to a rapid degradation. Structure and Function Analysis of Nop2p Analysis of the sites of point mutations in a protein can reveal the relationship among the protein primary sequence, conformation and function. To determine the changes in the primary structure of the mutant Nop2p, all of the six alleles were sequenced, and mutation sites were located (Table 7). Most of the point mutations caused amino acid substitutions (missense mutations) (Figure 19), while some mutations did not change amino acid residues (silent mutations) (Table 7). Only one point mutation resulted in a non-sense mutation (K605 to amber mutation in nop2-5) (Figure 19). Muhlrad et al. reported that the most common base changes are A/T>G/C transitions (Muhlrad et al., 1992). In this study, A/T>G/C and G/OA/T transitions comprised 57% of the total base changes in the six nop2 alleles, with G/C>A/T the most common change (33%) (Table 8). Each of the six alleles contained from 7 to 12 missense mutations. In nop2-10, all of seven point mutations were located in the C-terminal half of the protein sequence (from aa 254 to aa 560). In contrast, the mutations in all the other five alleles spanned the whole protein sequence. According to the sequencing data, replacement of some amino acid residues caused changes in charge or polarity at these sites. For example, I309K change in nop2-3 made this site become positively charged (Figure 19). Protein secondary and tertiary structures depend on the non-covalent interactions among the side chains of amino acid residues. Therefore, it is likely that the amino acid substitutions in the mutant Nop2p make the functional conformation of the protein unstable or disrupted at the restrictive conditions.

PAGE 95

88 Table 7. PCR-Generated Point Mutations in NOP2 Alleles Mutationst no P 2-3 Ai4 5 >G, Ti 97 >A*, G 53 1>A, A 778 >G, T 902 >A*, T 925 >A, T 9 4 >C*, Ano3>T, Ci202>T*, Gi2 9 i>A, Gi 40 8>A, Ai 640 >C nop2-4 G 20 7>A, A 25 2>G, A 25 6>T, A 286 >T, G 427 >T, G 520 >A, T 729 >C, A 829 >G, C 83 9>T*, G 9 is>A*, T 104 5>C, Gn 31 >A, Ci 489 >T nop2-5 G !92 >A, G 56 i>A, A 826 >G, Ci 6 2 >A, Tn 93 >C*, Gn 31 >A, Ai396>T, Gi 647 >A, Ai 749 >C, G 1758 >T, Ai 812 >T # nop2-6 a 42>t> a 322 >T, G 437 >A, G 465 >A, T 594 >A*, A 6 i 7 >G*, T 720 >A, T 792 >A, CA 944 >TT, Ci 00 5>T*, T 1319 >A, A 1450 >T, Cl547>T*, Gi 7 i 8 >T* nop2-9 A 86 >G*, AA 247 >TT, C 350 >T*, T 368 >G, A 450 >G, T473>A*, G 504 >A, T 520 >C, Cii 62 >A, Gii 74 >A, Ci 347 >T, Ai 529 >T no P 2-10 T 692 >C*, A 725 >T*, T 759 >A, G 898 >A, C 943 >A, G 972 >T, Ano7>T, Ti 292 >C*, Gi 494 >A, Ci675>A Numbers indicate positions of missense mutations in NOP2 sequence. Silent mutations Nonsense mutation

PAGE 96

89 Table 8. Types of Base Changes in the Six nop! Alleles Type of base change % observed Transitions AyT>G/C 24% G/C>A/T 33% Transversions A/r>T/A 29.3% A/T>C/G 0.26% G/C>T/A 11.6% G/C>C/G

PAGE 97

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PAGE 98

91 C CO CM a o c (0 a> U) c a 'o < o c E < CO E £ CO D D D o CO CD O O CD cn =3LO lo I LO _Q O b CO -•< v r* CO CO a -=r co LO i— -*<. O cn ld CD ->-co CO r ID O CD If) O o LO CD ar o C3-+-0 co oo CD CD o o CO CD co CO CO CO co co o CO CO CO rco ID LD co O cn CM CO CD CM CD CO 00 co co CD CD =3" O o co CM C3 CM sr CM CO rv CM co CO CO CD CM o r^ CO CO cn CM CO C_> ID CO a o o CO co CM O o CM CM > — >-z CD CD O O CO r-rco <--co CO o LU-*-> CD oo LO oo CC-*-C3 CD sr o CO t to CM a o c CM Q. c CM Q. O c CO CO B0 LO CD 3" CO o CD co CO CO CM Z--i^ CO CO c LO CD ^ CO I CM c LO CO i CM a o c i c

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92 Since the random mutagenesis PCR method was used in this study, it was predictable that multiple mutations spanning the whole protein sequence would occur. This was confirmed by the results from sequencing of the six nop2 alleles (Table 7). In many cases, Ts phenotype is caused by one or a few point mutations at critical sites ("hot spots"). To determine the mutations responsible for the phenotype, several restriction sites within the ORF of each nop2 allele was used to separate the mutations in each allele into several groups. The phenotypic effects of each group or the combination of several groups of mutations were analyzed. nop2-3 allele. In this allele, the two point mutations within Mfel fragment are not located in any of the motifs (Figure 19), and did not cause the phenotype (Figure 20, nop2-3a). Another two point mutations located 3' to Spel site were not required for the phenotype (Figure 20, nop2-3c). nop2-4 allele. In this allele, one Bsgl and two Mfel sites were used to separate the allele into four parts. The effect of the mutations in each part was tested. The six point mutations upstream to Bsgl site did not cause any detectable phenotype at 37C (Figure 20, nop2-4a). However, the six point mutations downstream to Bsgl site caused the Ts phenotype (Figure 20, nop2-4b). These six point mutations were then separated into three groups. Each group independently or the combination of the two groups did not cause the Ts phenotype (Figure 20, nop2-4c, nop2-4d, nop2-4g and nop2-4h). However, the effect of each group seemed to be additive (Figure 20, nop2-4g). The mutation in the SAM binding domain (I304T) (Figure 19) with another mutation in the Mfel fragment did not cause the Ts phenotype (Figure 20, nop2-4d). The results suggest that at least the three point mutations located in the three regions of Nop2p are required to cause the phenotype. Mutation in one of them may not significantly change the conformation. nop2-5 allele. The two point mutations in Mfel fragment of this allele caused the phenotype, though this allele was a little leaky (Figure 20, nop2-5a). Since A378T also occurs in nop2-4 but did not cause Ts phenotype, it may be A355 to D change that caused

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Figure 20. Mapping of the mutations in the six nop2 alleles. The point mutations (labeled as asterisk in boxes) in each of the six nop2 alleles were separated into groups using the restriction sites Bsgl, Mfel, orSpel (see METHODS AND MATERIALS for detailed descriptions). The nop2 alleles thus constructed are shown as the boxes (the left panel). Serial dilutions of the cells carrying the corresponding nop2 alleles were grown on the SD plates at 25C (the middle panel) or 37C (the right panel). YBH31 strain carrying the plasmid-borne NOP2 (labeled as NOP2) is shown as a control.

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94 Bsgl Mfel Mfel Spel K J_ J L IN 1 • 1 CI IN > > 1 t ** CI IN \*> CI |N : * 1 L| IN LI |N 1 hh|*u | i i 1 ; L| IN * i 1 CI IN I i CI IN 1*1 1 CI |N I 1 LI IN \ CI IN 1 ^ 1 CI IN LI IN 1 1 1 1 CI IN | : 1 L| IN 1 1 ^CI |N b***CI IN | 1 CI |N L] |N 1 l 1 U |IN | ** i 1 LI IN • • 1 1 CI IN 1 • 1 CI IN CI CJ N0P2 nop2-3a nop2-3b nop2-3c nop2-3d N0P2 nop2-4 nop2-4a nop2-4b nop2-4c nop2-4d nop2-4g nop2-4h N0P2 N0P2 nop2-5a nop2-5b nop2-5c nop2-5d N0P2 nop2-6a nop2-9a nop2-9b nop2-10a N0P2 25C • ••• • /• .' • # if • A £ 37C • f • • • • # % •• % r # • • # 4i 4 • • • • # .; • • • • ^ • tr r .. • • ••* • • • ft • • t • • •••

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95 the Ts phenotype (Figure 19). This change makes this site negatively charged, which is located in the SAM binding domain (Figure 19). However, it is not clear if the phenotype was caused by inactivation of the methylase activity or by disruption of the functional conformation. However, without these two point mutations, the other point mutations in this allele also conferred the cells with the Ts phenotype (Figure 20, nop2-5b and nop25d), suggesting that at least two structural domains present in Nop2p and that the mutation in either domain causes disruption of the functional conformation. Truncation of 14 amino acid residues at the C terminus caused by a nonsense mutation (K605 to amber mutation) did not significantly affect the function (Figure 20, nop2-5c). The three point mutations downstream to Spel site did not cause Ts phenotype (Figure 20, nop2-5c). nop2-6 allele. One Mfel site was lost by mutation in this allele. The three point mutations in the BsgVMfel fragment were not responsible for Ts phenotype (Figure 20, nop2-6a). nop2-9 allele. In this allele, the three point mutations within the Mfel fragment caused the phenotype (Figure 20, nop2-9a). However, all of the three sites are outside the conserved motifs (Figure 19). The other point mutations in this allele did not exhibit any phenotype (Figure 20, nop2-9b). nop2-10 allele. Only the five point mutations in BsgVMfel fragment were responsible for the Ts phenotype (Figure 20, nop2-10a). This allele caused both Ts and Fs phenotypes. It is not known if the two phenotypes can be separated. However, it is possible that at least two structural domains exist in BsgVMfel fragment. Mutation in one domain may cause one phenotype but not the other. In summary, there is no "hot spot" mutation site in Nop2p. The amino acid substitutions in the six alleles are spread over the whole protein sequence. The A355D change alone (in nop2-5) seems to cause a "leaky" Ts phenotype. However, in all of the other alleles, at least three point mutations are required for causing the Ts phenotype. This suggests that there may be several structural domains within Nop2p. Mutation in more

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96 than one structural domains will cause a significant conformation change under the restrictive conditions. Nop2p and Methylation Nop2p Contains A Putative SAM Binding Domain A further study was done in order to understand the detailed mechanism by which Nop2p is required for pre-rRNA processing. Proteins with their functions essential for cell viability (encoded by "house-keeping" genes) usually contain evolutionarily conserved sequences (domains or motifs). Therefore, analysis of the primary structure of a protein will give a clue to its function. Koonin found that human pi 20 protein as well as E. coli. Fmuv protein contains the consensus sequence, the SAM binding domain, which is conserved in a broad spectrum of methylases that modify proteins, nucleic acids, and lipid substrates (Koonin, 1994). The function of Fmuv is not known, but it is encoded in the same operon with fmt, the Met-tRNAfMet transformylase (Meinnel et al., 1993). Three other motifs are also present in pi 20 and Fmuv proteins. Since Nop2p is highly homologous to the pl20 protein (de Beus et al., 1994), the possibility that Nop2p might contain SAM binding domain was investigated. Sequence alignments revealed that Nop2p also contained the SAM binding domain and the other two motifs (Figure 21). This result suggests that Nop2p may have methylase activity. Since Nop2p is required for processing of 27SB to 25S rRNA, it is possible that Nop2p might be involved in the methylation at the 27S level (late methylation), and that the methylation might be required for a correct secondary structure of 27S pre-rRNA for processing. Among the methylations in 25S rRNA sequence, only four groups are added as late methylation (at 27S level) (Brand et al., 1977). Two of them are contained in a stretch of sequence (UmGm l FUC2922), which is evolutionarily conserved from bacteria to human (Table 9).

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97 Table 9. Conserved UmGm^UC in the Large Subunit rRNA Sequences Species LSU rRNA Sequence and site of ribose methyl ation Total number of ribose methylations E.coli 23S C 2 55iUmGUUCG 3 Yeast (Mito) 21S C 2 79oUmGUUCG 2 Yeast 25S U 2 9i7UmGm x FUCA 37 Xenopus 28S U 36 24UmGm v FUCA 70 Human 28S U 4 45 7 UmGm t FUCA 70 LSU: large subunit; Mito: mitochondria; 4 1 : pseudouridine.

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Figure 21. Multiple sequence alignment of yeast Nop2p, human p 1 20, and E. coli Fmuv proteins. The alignment was generated by using MacDNASIS software. Amino acid numbers are indicated in parentheses. The positions of the SAM-binding motif, the motif 2 and motif 3 are indicated. Con: consensus sequence.

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99 Motif 1 i— SAM-Binding — 1 Nop2 (330) QAASSFLPVIALDPHENERILDMAAAPGGKTTYISAMMKNTGCVFANDANKS P120 (369) QGASSMLPVMALAPQEHERILDMCCAPGGKTSYMAQLMKNTGVILANDANAE Fmuv (231) QDASAQGCMTWLAPQNGEHILDLCAAPGGKTTHILEVA-PEAQVVAVDIDEQ Con Q. AS. a. L. P. E. ILDn. APGGKT. a. dd noA. Da. Motif 2 Nop2 RTKSLIANIHRLGCTNTIVCNYDAR-EFPKVIGGFDRILLDAPCSGTGVIGKDQSVKV P120 RLKSVVGNLHRLGVTNTIISHYDGR-QFPKVVGGFDRVLLDAPCSGTGVISKDPAVKT Fmuv RLSRVYDNLKRLGMKATVKQGDGRYPSQWCGEQQFDRILLDAPCSATGVIRRHPDIKW Con R. . No. RLG. To FDRoLLDAPCS. TGVI. +. nK. Motif 3 Nop2 SRTEKDFIQIPHLQKQLLLSAIDSVDCNSKHGGVIVYSTCSVAVEEDEAVI (489) P120 NKDEKDILRCAHLQKELLLSAIDSVNATSKTGGYLVYCTCSITVEENEWVV (528) Fmuv LRRDRDIPELAQLQSEIL DAIWPHLKTGGTLVYATCSVLPEENSPAD (386) Con + Da LQ. nL D. a K. GG. dVY. TCSn. EE. dd a = A. F. I. L. M. V + = K. R = D. E

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100 Level of Methylation at the Conserved UmGm v FUC2922 Site was Reduced during Nop2p Depletion To investigate if Nop2p was involved in the methylation at the conserved UmGm v FUC2922 si te > an RNase protection assay was designed to detect any effect of Nop2p depletion on the methylation at this site. The cellular RNAs in YBH5 cells depleted of Nop2p for 8.5 hours and the wild type cells (L4717) were first pulse labeled with [me^y/3 H]methionine and chased with an excess of methionine. Oligonucleotide (oligo) 7 was used to protect the UmGm v FUC2922 site (Figure 22A). Oligo 8, used as a control, would protect two early methylation sites in the sequence immediately following that protected by oligo 7 (Figure 22A). Oligo 6, used as an internal reference control, would protect the three early methylation sites located at a significant distance from the UmGm4 / UC 2 922 site. In Nop2p-depleted cells, the intensity of the band protected by oligo 7 was significantly reduced compared with that in the wild type L4717 cells (Figure 22B). To evaluate the efficiency of isotope incorporation, equivalent amounts of the labeled RNAs from L4717 and from the Nop2p-depleted (YBH5) cells were partially digested with RNaseTl Nop2p-depletion caused a reduction in the incorporation of [methyl3 H]methionine label into the RNAs (Figure 22C). Even considering this factor, comparison of the intensities of the bands protected by oligo 7 and oligo 8 (both covering two methylation sites) in YBH5 revealed that the intensity of the band protected by oligo 7 was reduced to a much greater degree (Figure 22B). To correct for this difference in the efficiency of isotope incorporation, the radioactivity in the protected bands was measured by scintillation counting. The data from YBH5 were normalized by using the amount of radioactivity protected by the control oligo 6. In three independent experiments, the oligo 7-protected signal from YBH5 was only 66% (3%) of the control signal from L4717. In

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T3 t/T § (U 4— "3 X. T3 — o u 1 3 a o E 03 OJ y U E X. c/5 O y T3 1) to H c^ in 3 •a 3 -*""* U -t— 1 00 c x: 4— < x. o u. y a _>> 4— < o j3 c s o -t— *— > c 00 *4-t o u *o 3 C o E 3 u 4-j y f3 ^^ o 13 OJ E 3 c3 '3 E a. c _o *4-* o u 4-J c au E u la E Q 00 y 3 C I OJ I 11 C o E Oh U 3] 03 Z E .2? ^c O 60 O c3 z s c u o c o u —J uif U o E <2 c U *-> *B U E £ T3 *— 1 o _o o 3 c M "o < c 3 cr y -a X o £ U -C 4-1 o 4-* 9) C i l t— l -C 'i c 71 N fa 'C o a. 73 C "3 U > o ;/: OJ u 00 r3 u y 3 00 U "ft E a 3 y •y. 3 < 4—* o 4— X. >— • (O 4^ 03 E u. 03 — W o 3 x: 00 rad Ui < aj *-3 y E OJ T3 1 O *-< — i c ts o o o 3 > -a 3 5 3 < 4-J V O H •— X) E 3 '•3 S3 5 E c u E u Oh E o c o C3 ft, | C E m a u a E E a y -3 u u x: *— i '4Q "3 u E fd IS — -4-* •3 U u Xj r-~ c o c r3 Q o c 3 oc T3 *—> O 3 o IE a, c 3 1) x: 3 '3 C c E jr o 4— CA 2 03 o u a C/3 C/3 a a u Q H E c o T3 3 3 — c c 4-^ eg ID T3 G 1 o c u 3 cr SB i H X) < c D 13 > '3 y E x: r3 *s y x: 3 a 3 O m CO o — J, u 3! [2 o 4—1 o -a -a a a c o "o u g u 73 JS OJ OS '5 x: Oh 03 C ID 3 03 OJ 4— 1 03 it 03 OU i/i c o o 3 B u o 1 1 y -o *-* U So o E O 3 H 2 2 1/3 i; u 3 o '7. o a. C E o c 3 cr 3 3 n J-. 73 IS x: *-^ s x: '5 E

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102 SH9A LHP! IB (0 — z O SH9A o o o SH9A 9H9A SH9A I 1 m 0Q

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103 Table 10. RNase Protection of [3H]-Methylated rRNA Oligo 6 Protection Oligo 7 Protection Oligo 8 Protection Experiment L4717 DPM Factor % L4717 YBH5 DPM DPM % L4717 YBH5 DPM DPM % 1 3929 2.57 100 2163 1522 70 3124 3058 98 2 1423 3.27 100 723 471 65 716 699 98 3 975 2.19 100 334 214 64 325 307 95 Mean 66.3 97.0 Standard Deviation (S n -1) 3.2 1.7 Oligonucleotide 6 was used as an internal standard. YBH5 DPM data for each experiment were multiplied by a correction factor in order to normalize to 100%.

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104 contrast, the signal of oligo 8-protected band from YBH5 was 97% (1 .7%) of that from L47 17 (Table 10). There are two explanations for the 33%, but not 100%, reduction of methylation at UmGm4 / UC2922 site. One is that Nop2p may be responsible for only one of the two methylations. Another explanation is that another methylase may also modify these sites, but with less efficiency. In summary, the methylation at UmGm x FUC2922 site was significantly reduced during Nop2p depletion. However, it is not clear about the relationship between the reduced methylation and the defective processing, both of which occurred almost at the same time during Nop2p depletion. They may be only temporally related or may have a cause-effect relationship. Analysis of Methylation by Using Primer Extension Assay To understand more about the relationship between methylation and Nop2p function, primer extension analysis was used. It was found that during reverse transcription, reverse transcriptase may "pause" at a site modified by 2'-0-methylation on an RNA template if the concentration of dNTPs are low (concentration dependent pause) (Maden et al., 1995). To test this technique, RNAs from the L4717 cells and the in vitro 27S rDNA transcript (with no modification) were used for primer extension. Concentration dependent pauses at the sites modified by base methylation (mU284u) and 2O' -methylation (UmGm29i9 and AmGCm2945) were observed with the RNAs from L4717, but not with the in vitro transcript (Figure 23). This result indicates that with this assay, a 2'-0-methylated site or a base methylated site can be distinguished from a nonmethylated one. A few pauses were also seen with the in vitro transcript (Figure 23), which may be caused by RNA secondary structures. To evaluate the effect of Nop2p depletion on rRNA methylation, total RNAs from the cells depleted of Nop2p for 8.5 hours

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Figure 23. Analysis of rRNA methylation by using concentration dependent pause assay. The RNAs from L4717 and YBH5 cells grown in YPD medium for 8.5 hours were used as templates. In vitro 27S rDNA transcript was included as a negative control. The templates were mixed with a primer JA22, which was extended in the presence of [a35 S]dATP by reverse transcriptase to the methylation sites as indicated. Serial five-fold reductions in dNTP concentrations (500 to 4 |iM) for extension are indicated.

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106 In vitro L4717 o o o o o o o o o o iot-cvjtjm •c\j <* • --h YBH5 o o o o o lo^-ovjTtGATC •— = : = = I % z • "x ^2842 S' m .mJ t : i A U Urn Gm U C 2922 Z _r G • J Am • G Cm • -,U • T G2 947 i

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107 were subject to primer extension analysis with a primer that is extended from G2969 to UmGm v PUC2922However, a pause was still observed at UmGm4 / UC2922 site after Nop2p depletion, which suggests that the site was still modified (Figure 23). The mU2840 and AmGCm2945 sites were also modified during Nop2p depletion (Figure 23). The effect of the mutation in nop2 on the methylation was also analyzed. Two Ts strains were tested. nop2-4, with a tight Ts phenotype, was incubated at 37C for 2 hours, the time point when an obviously abnormal polysome profile was already observed (see Figure 17). nop2-6, with a leaky Ts phenotype, was also tested with 2 or 24 hours of incubation at 37C. However, in both strains under the restrictive condition, the concentration dependent pause bands were seen with no difference from the control YBH31 (not shown). Considering the possibility that Nop2p might be involved in methylation other than the UmGm v FUC2922 site, more primers were used to analyze all of the mapped methylation sites on 25S rRNA sequence (Table 11). Nevertheless, no detectable reduction of methylation was seen in the two Ts strains at the restrictive condition (not shown). One explanation for this result is that Nop2p might not be involved in methylation, which is inconsistent with the result from RNase protection experiment. Another one is that a slow methylation at UmGm4 / UC2922 site in Nop2p depletion or Ts cells still occurred and that mature rRNAs have a long half-life, so that in a mixture of methylated and non-methylated RNA templates, the difference could not be differentiated by concentration dependent assay. One possible solution to this problem is to use 27S intermediate, instead of total RNAs, for primer extension. However, because of very low level of 27S and no 27S band visible on the gel stained with ethidium bromide, efforts to gel-isolate 27S for primer extension were made twice without success.

PAGE 115

108 Table 11. Methylation Sites of 25S rRNA and Primers Used for Concentration Dependent Pause Assay Methylation sites Primers* mA643, AmCm648 E GmAAmgos, Amgis, Gmg65, Umg96 D Ami i3i C Cmi435, AmGmi448 B Umiggs A mA2i40, Cm2i95> Am22i7, Am2254, mCAAmAm2279 JA31 Um24l5> rnU2632> Am2638 JA30 Um2725 GmAGm2788> mU2838, UmGm29i9, AmGCm2943 JA22 : see Table 4 for the primer sequences.

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109 Interaction of Nop2p with Other Macromolecules Immunoprecipitation Approach Recent studies suggest that certain nucleolar proteins are associated with snoRNAs as a functional complex (Fournier and Maxwell, 1993). Many yeast nucleolar proteins identified so far contain recognizable RNA binding motifs, such as RRM, GAR domain. Nop2p does not contain any of the known RNA binding motifs. However, in several cases, proteins without these motifs were also found to be associated RNA. One example is the LI protein, a component of large ribosomal subunit. LI protein does not contain any of these motifs, but it was found to be associated with 5S rRNA. It was suggested that some arginine residues in the protein be involved in RNA binding (Deshmukh et al., 1993). Therefore, it was still possible that Nop2p might be a component of snoRNP by a direct interaction with snoRNAs. Another possibility is that Nop2p might be recruited by snoRNPs through protein-protein interactions. To investigate the possibility of a protein-protein interaction, immunoprecipitation (IP) was done by using the anti-Nop2p antibodies. In order to increase a chance of detecting the interaction, one monoclonal and one polyclonal antibodies against Nop2p were tested. Since Noplp is known to be associated with several nucleolar proteins and with at least the 1 1 yeast snoRNAs (Schimmang et al., 1989), an anti-Noplp antibody was used as a positive control. [ 35 S] labeled cellular proteins from L4717 cells were precipitated by the antibodies separately, and analyzed with SDS gel electrophoresis. One band at the position corresponding to that of Nop2p was seen in the precipitates with the two anti-Nop2p antibodies, but not with the negative control (without any antibody) (Figure 24A). This suggests that Nop2p was specifically precipitated by the anti-Nop2p antibodies. However, compared with the negative control, no extra band was seen in the precipitates with the two anti-Nop2p antibodies (Figure 24A), suggesting that there was no

PAGE 117

o — <*-> 3 i£ S a o o eg u u o O 0 > c C 1 w fi u Q. O C 3 3 o X U 173 > O c 3 z i *3 *j o C a -o c 'S O I. c/3 a a A 8 .5 3 ^5 jU 13 X) 3 U E C/3 3 X Oh _o i -a 1 g 3 > T3 O X '4— g eg u J-. o ueg X Oh u t/5 Oh . T3 O X •t— C C3 'I X > "o a, en x < Oh Oh < (N Oh O Z O < Oh l 00 Q oo X 1) 4-H 3 C O U D o 2 > h9 x '5 u X b 03 Oh i CO c rt o ^ 2 1 p X O <-> x rH-J Is 03 --n QQ X w o ft "^ 03 o3 X O 1 1 O a i ,— E Cm £ Oh Z r03 X •o 03 u. 03 Oh 1) n -a S 03 "S ^ 8 w x I) o H-^ 03 X Oh C/3 o X Oh X3 u X S E U =5 SB < z S T3 CO h9 03 E o S u x c o i 'a o CJ E O. o B 3 E 4J 60 3 _>. O O. I 03 O — 3 3 S x T3
PAGE 118

Ill o (ftpoqi^uv on) lo-nuoo (dzdoN-UUV) £qvddV (dZdoN-uuv) 89C (didoN-uuv) 99V I I I I m (fipoquuvoN) Ioj^uoq (dZdoN-uuv) £qv
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112 proteins specifically associated with Nop2p. Considering that the protein-protein interaction might be broken by the in vitro treatment, different buffer conditions with a low or high ion concentration were tested. In addition to the whole cell lysate, nuclear homogenate was also used for IP. However, all gave a similar result (not shown). Since in the positive control (anti-Noplp antibody), many proteins were found in the precipitate (Figure 24A), this indicates that Nop2p was not associated with any proteins under the conditions used here. Nevertheless, it can not be excluded that in vivo Nop2p may interact with other proteins through a weak, transient association. To determine if Nop2p might be associated with snoRNAs, the wild type L4717 cells were labeled with 32 P-phosphorus. The whole cell lysate (Figure 24B) and the nuclear preparation (Figure 24C) were used for IP as described above, and the labeled PvNAs in the precipitates were extracted and analyzed. Many bands were seen in the precipitate with anti-Noplp, suggesting that Noplp was associated with many PvNAs (Figure 24B and C). This is consistent with the observations made by other researchers (Schimmang et al., 1989). In the negative control, several bands were also seen, which may be caused by a non-specific binding (Figure 24B and C). Compared with the negative control, no extra band was seen in the precipitates with the two anti-Nop2p antibodies (Figure 24B and C). Considering that no known RNA binding sequence is present in Nop2p, Nop2p may not have a direct association with RNA. Genetic Approach Analysis of macromolecule interaction by IP can only reveal those physical interaction that can endure in vitro treatment. A weak, transient, but functional interaction can be missed by this approach. The advantage of genetic approach is that it can reveal in vivo functional interaction. Reduced affinity caused by mutation in one protein may be complemented by overexpression of another interacting protein. A functional deficit caused

PAGE 120

113 by mutation in one protein may be compensated for by a high level of another protein with homologous function (Huffaker et al., 1987). These are the bases for one genetic method used here, that is, high-copy suppressor screening. A yeast genomic library carried on a high-copy 2 |im plasmid was transformed into all of the six nop2 Ts strains. The number of colonies screened for each Ts strain was enough to cover the whole library (Table 12). About 100 plasmids were recovered from the colonies formed at 37C. Most of the plasmids were different from the library plasmids with a lower molecular weight at ~5-kb, as compared with 6.9-kb of the library plasmid backbone (not including the inserts). The plasmids carrying the nop2 Ts alleles were also isolated. These nop! alleles were present in the strains used for the screening. No NOP2 gene carried on the library plasmid was isolated. This unexpected result is most possibly due to the way of preparing the library. This library was prepared by Sau3A partial digestion of yeast genome (R. Henriquez, personal communication). Since in NOP2 sequence, there are 10 Sau3A sites, it is quite possible that NOP2 gene might be destroyed during the preparation process. Only one library plasmid with a 3.7-kb insert was isolated because it improved, though very little, the growth of nop210 strain at 37C (not shown). The subcloned insert exhibited a very weak suppression effect on the phenotype of nop210 strain (not shown). Sequencing of the insert revealed that it was part of rDNA gene, ranging from the middle of 1 8S to the middle of 25S rRNA sequences (Figure 25). Since the suppression effect was extremely weak and no protein ORF found in the insert, it may not be a specific extragenic suppressor.

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114 Table 12. Screening for High-Copy Suppressors nop2 allele (imants screem sd Candidates Suppressors 5700 4 7900 3 1600 2000 50 1320 2 4900 50 1 nop2-3 nop2-4 nop2-5 nop2-6 nop2-9 nop2-10

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T3 1a bfl a "9 00 oq v, 'h— > 00 C 'oa oo o c 3 H3 -o XI en a Oh E u o -C y; ~, *— • o o o o -— it H— 1 ^ O >-. H— a •a 3 cr tv OJ 4— aj *> 5 c3 u H x "9 X C v: c +-H 3 JD M X c o 5 Q "3 a M u — i 3 C3 M a rM Oh > Oh O U OJ Oh o 0

PAGE 123

116 CO LO C\J C\J CO o 00 (N ON 00 m CO 00 CO s g. CO CO 00 Q o en CO LO CO

PAGE 124

DISCUSSION Nop2p is Essential for Cell Viability and Growth Gene disruption in diploid strains, followed by sporulation to test the viability of haploid segregants is thought to be the least biased technique for defining essential genes (Olson, 1991). Previously, it was demonstrated that replacement of 755 bp of NOP2 ORF with URA3 caused a lethal phenotype (de Beus et al., 1994). In order to eliminate the possibility of recombination between the chromosome and the complementing plasmid, another gene disruption was made. In this study, over 92% of the NOP2 ORF in five diploid strains was disrupted and replaced with either a HIS3 or a LEU2 marker gene. However, after sporulation and tetrad dissection, all of the four spores from the same ascus were viable with 2:2 segregation for the marker gene. This raised a question about if the gene was essential. However, Nop2p was detected in all of the spores and an intact copy of NOP2 was present in all of the spores with two of the spores carrying an additional disrupted copy of NOP2. This is not a strain-related phenomenon, since five diploid strains were tested with the same result. Apparently, a rare gene duplication event occurred during sporulation stage. During the process of this work, a similar observation was reported (Santos and Ballesta, 1994). When disruption of a gene encoding an essential ribosomal protein, PO, was made in a diploid strain, a possible sporulation-related gene duplication event was also observed (Santos and Ballesta, 1994). Since all of the viable spores carried one copy of NOP2 gene and expressed it, this phenomenon actually suggests that the gene is required for cell viability. When gene disruption was made in a haploid strain, therefore eliminating the sporulation stage, the viability of the NOP2117

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118 disrupted haploid strain was dependent upon the plasmid-carrying NOP2 gene. This result supports that the gene is essential. Nop2p function is required for cell growth. During first 8 hours of Nop2p depletion, the cell growth was not affected. This is because the cells contained a high level of Nop2p overexpressed during the pre-growth in YPGal medium. However, further depletion caused a progressive impairment of cell growth. Over 90% of the cells depleted of Nop2p for 28 hours were still viable. This is probably due to a basal activity of GAL10 promoter even in the presence of glucose repressor, a phenomenon also observed in the other depletion studies (Moritz et al., 1990; Deshmukh et al., 1993). Stronger evidence is from the mutational analysis of NOP2 gene. All of the six Ts strains containing the mutant protein were dead when incubated at 37C, but the lethal phenotype was rescued by a plasmid-borne NOP2 gene. These results confirm that NOP2 gene is indispensable for cell viability and growth. Nop2p is Required for Ribosome Synthesis The fact that NOP2 gene is essential for cell viability and growth indicates its important cellular functions. Depletion of Nop2p caused a significant reduction of the 60S ribosomal subunit but a relative accumulation of the 40S subunit. A relatively specific feature associated with the shortage of 60S subunit is the halfmer polysome, which was observed during depletion of the large subunit protein LI or LI 6, and during depletion of Nop4p/Nop77p, a nucleolar protein involved in the synthesis of 60S subunit (Moritz et al., 1990; Deshmukh et al., 1993; Berges et al., 1994). The cells depleted of Nop2p clearly exhibited this feature. Since the reduction of 60S subunit level was observed before the cell growth slowed down, the defect was directly caused by Nop2p depletion. A more convincing evidence is from the mutational analysis of the protein. All of the mutant protein generated at the restrictive condition will be almost immediately

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119 inactivated, therefore eliminating possible secondary effects. In all of the six nop2 Ts strains, two hours of incubation at 37C caused a significant defect in the synthesis of 60S subunit. Some of the Ts strains exhibited an abnormal polysome profile even under permissive conditions, suggesting that the severe mutations affected the protein conformation even at the permissive condition. In the nop2-10 strain, pre-rRNA processing at 25C is similar to that in the control strain. However, at this temperature, a defect in the synthesis of the 60S ribosomal subunit was obvious, which suggests that Nop2p may be involved in the other steps of ribosome synthesis as well as in pre-rRNA processing. The results from the depletion and mutation studies of Nop2p clearly indicate that Nop2p is essential for synthesis of 60S ribosomal subunit. Synthesis of ribosome involves multiple steps, including transcription of rDNA gene, processing and modification of pre-rRNA, expression of ribosomal proteins, assembly of rRNAs with ribosomal proteins, and export of ribosomal subunits to the cytoplasm. Nop2p is Required for Pre-rRNA Processing rDNA is transcribed as a single large pre-rRNA, which contains the sequences of rRNAs. Processing of the pre-rRNA into mature rRNAs is an important event in ribosome synthesis. Since a general outline of processing of pre-rRNA has been known, pulse labeling of pre-rRNA and chasing it to mature rRNAs will reveal a defect in the processing. The processing of pre-rRNA in the wild type L4717 was finished within 5 minutes, as was expected (Girard et al., 1992; Russell and Tollervey, 1992). Depletion of, and mutation in Nop2p caused a significant reduction in the formation of 25S rRNA, but the processing to 18S rRNA was not significantly affected. This suggests that Nop2p is involved in the step after the separation of 18S and 25S pathways. Analysis of the accumulated processing intermediates revealed a significant accumulation of 27S intermediate in Nop2p-depleted

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120 cells and in several of the Ts strains (nop2-3, nop2-6, nop2-9, and nop2-10). Analysis of the steady-state levels of the processing intermediates indicates that both 27SA and 27SB intermediates significantly accumulated in the Nop2p-depleted cells. This suggests that the defect caused by the deficit in Nop2p function was at the step of conversion of 27SB into 25S rRNA and 7S, a direct precursor of 5.8S rRNA. Reduction in 5.8S rRNA during Nop2p depletion supports this conclusion. Based on these results, we conclude that Nop2p is required for the processing in ITS2, probably at C2 site (see Figure 10). Several yeast nucleolar proteins have also been shown to be required for the formation 25S rRNA. However, depletion of them separately exhibited different features from depletion of Nop2p, and they are required for processing at the sites different from the one requiring Nop2p. Nop4p/Nop77p was found to be involved in the processing pathway to 25S rRNA. However, depletion of Nop4p/Nop77p caused a significant reduction in the 27SB intermediate, which suggests that it may function at the 27SA level within 3' half of ITS1 (Berges et al., 1994; Sun and Woolford, 1994). Nop3p, though localized in the nucleus, was also found to be required for the processing of 27SB to 25S rRNA (Russell and Tollervey, 1992). However, this protein is also involved in the processing of 20S intermediate to 18S rRNA, the only processing step occurring in the cytoplasm. Depletion of Nop3p for a long period of time revealed the accumulation of both 23S and 27SB processing products (Russell and Tollervey, 1992), which arise from cleavage of the 35S primary transcript at or near site B\ without prior cleavages at A\ and A2 sites. This suggests that Nop3p is possibly required for the processing in ITS 1 Dbp3p is required for the processing at A3 site in ITS 1 Depletion of Dbp3p caused an accumulation of 27SA but a reduction of 27SB intermediate (Weaver et al., 1997). In the yeast rrpl Ts mutant, the 27S intermediate was rapidly degraded (Fabian and Hopper, 1987). Since ribosome assembly and processing of pre-rRNA occurs simultaneously, a defect in the assembly may affect the processing. Temperature-sensitive mutations in LI 6, a protein component of 60S subunit, caused a defect in the processing of 27SB to 25S

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121 rRNA. However, the 27SB and other processing intermediates were rapidly degraded (Moritz et al., 1991). Therefore, Nop2p is probably the first of the nucleolar proteins identified so far, which is required for processing in ITS2. Disappearance of the 27S intermediate in nop2-4 and nop2-5 strains is most possibly caused by a rapid degradation, since primer extension analysis of nop2-4 strain incubated at 37C for two hours did not detect any change in the methylation of the 27S sequence. These results suggest that Nop2p is involved in the stability of the 27S intermediate. Possible Roles of Nop2p in the Other Events of Ribosome Synthesis Nop2p and rDNA transcription. The role of Nop2p in the transcription of rDNA gene was evaluated by pulse-chase labeling with [ 3 H]uracil. In all of the six Ts strains and the cells depleted of Nop2p, a significant accumulation of 35S transcript, which is the largest recognizable precursor for rRNAs (Woolford and Warner, 1991), was observed, indicating that rDNA was still transcribed in Nop2p-depleted cells or in the nop2 Ts cells. This suggests that Nop2p is not involved in the transcription of rDNA gene. Nop2p is not a structural component of 60S ribosome subunit. Depletion of a protein component of 60S subunit will also reduce the level of the subunit and cause a rapid degradation of 25S rRNA and the other components of this subunit. Since Nop2p is mainly localized in the nucleolus (de Beus et al., 1994), it is not possibly a structural component of 60S ribosomal subunit. Depletion or mutation of Nop2p caused not only a reduction in 25S rRNA but also an accumulation of 35S 32S and 27S prerRNAs, suggesting that there was a delay in the pre-rRNA processing, and that the reduction of 25S rRNA was not caused by a rapid degradation due to disruption of 60S subunit. Nop2p and ribosome assembly. In prokaryotes, ribosome subunits can be assembled in vitro (Nomura, 1973). However, this has not be successful in eukaryotes at

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122 the current time, which reflects the complication and multiple factors required in eukaryotes. Therefore, currently, it is difficult to analyze the direct role of a nucleolar protein in the assembly process. Although Nop2p does not contain a GAR domain or an acidic domain, it can not be excluded that the acidic or basic residues in the protein sequence may facilitate ribosome assembly. It is also possible that Nop2p may modify ribosomal protein(s) or rRNAs, and therefore promotes the correct assembly of ribosomes. Nop2p and nucleolus-cytoplasmic transport. Nucleolin and B23 were found to shuttle between the nucleolus and the cytoplasm (Borer et al., 1989), suggesting that they may be involved in the transport between these two compartments. There is no direct evidence suggesting that Nop2p has such a function. Since a small portion of Nop2p was found to be in the cytoplasm (de Beus et al., 1994), it is possible that Nop2p may also shuttle between the nucleolus and the cytoplasm to help transport of the ribosomal components or the mature ribosome. Interaction of Nop2p with Other Macromolecules Intracellular macromolecules related to the same functional pathway usually interact physically and/or functionally with each other. Analysis of such interactions will help understand more about the protein function. Several approaches have been used in this study in order to detect any functional or physical association of Nop2p with other macromolecules. Immunoprecipitation was successfully used for analysis of association of Noplp, Garlp or Soflp with snoRNAs and nucleolar proteins (Schimmang et al., 1989; Girard et al., 1992; Jansen et al., 1993). However, no such association was found with Nop2p. Since Nop2p does not contain any of the known RNA binding motif, such as RRM or GAR domain, it may not directly associate with any RNAs. However, a weak proteinprotein association between Nop2p and other proteins may still exist. Such association

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123 may not survive the in vitro treatment. One piece of evidence supporting this assumption is from the mutational analysis of Nop2p. In nop2-4 and nop2-5 strains incubated at 37C, the 27S pre-rRNA was rapidly degraded, while in the other Ts stains and Nop2p-depleted cells this pre-rRNA significantly accumulated. This suggests that there may be a region in Nop2p that is related to the association of other nucleolar proteins with 27S pre-rRNA, and that the mutations in nop2-4 and nop2-5 alleles disrupt such association, resulting in a rapid degradation of this pre-rRNA. The yeast two hybrid system can detect intracellular proteinprotein interaction, and therefore is a more sensitive method than immunoprecipitation. Nevertheless, no protein interacting with Nop2p was isolated (Wu, P. and J. P. Aris, unpublished results). The search for high-copy suppressors of the nop2 alleles also gave a disappointing result. Although there have been several successful application of this genetic approach, the limitation is that suppressors isolated may not be functionally related to the protein of interest (bypass suppressors) because it aims for "gain of function" (Doye and Hurt, 1995). In this study, a piece of rDNA sequence isolated may belong to the "bypass suppressors". Another genetic approach, synthetic lethal screen, is based on "loss of function", and has been extremely successful (Doye and Hurt, 1995). Screening for the genes synthetic lethal with NOP2 alleles is currently under way in this lab. The Mechanism by Which Nop2p Functions Almost all of the yeast nucleolar proteins identified so far have been shown to be involved in processing of pre-rRNA. Only a few of them, such as Rntlp (RNase III), Xrnlp and Ratlp, were found to have direct nucleolytic activities (Stevens et al., 1991; Amberg et al., 1992; Elela et al., 1996). However, the molecular mechanisms for the functions of the other nucleolar proteins in pre-rRNA processing have not been clearly understood. Many nucleolar proteins contain a RNA binding motif, such as RRM or GAR domain. It can be imagined that some nucleolar proteins may bind to pre-rRNA to facilitate

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124 its folding, which is important for the processing. Since some nucleolar proteins were shown to be physically and functionally associated with snoRNAs, they may function as components of a snoRNP complex. One of the features about Nop2p is that there is no RRM or GAR domain present in its sequence (Aris, J. P. and B. Hong, unpublished results). Therefore, the underlying molecular mechanism for Nop2p may be different from those for the other nucleolar proteins. Nop2p and rRNA Methylation Proteins with their functions essential for viability are usually evolutionarily conserved. Comparison of protein sequences will reveal conserved motifs and offer a clue to the protein function. Protein sequence alignments indicates that Nop2p contains the SAM binding domain, which is conserved in methyltransferases. Since rRNAs are greatly modified with methylation, this raises an interesting possibility that Nop2p may have a rRNA methylase activity. During Nop2p depletion, the methylation at the UmGm4 / UC2922 site in 25S rRNA sequence was reduced. The methylation at this site has been mapped at the 27S intermediate level (Brand et al., 1977), and processing of this intermediate to 25S rRNA may require Nop2p function. Therefore, the methylation and the processing is clearly related. Since this stretch of sequence and the methylation sites are evolutionarily conserved from bacteria to human, it is very possible that the methylation at this site is essential for ribosome synthesis and ribosome function. Methylation increases local hydrophobicity, and therefore may help to form the secondary structure of pre-rRNA, which may be required for the processing or for the binding of ribosomal proteins to 27S intermediate. If this is true, then the methylation is required for the processing. Such relationship was observed in the cultured mammalian cells. When deprived of methionine or treated with an inhibitor of methyl transfer, the cells had a defect in the processing of pre-rRNA

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125 (Swann et al., 1975; Wolf and Schlessinger, 1977). However, to prove the causal relationship, it has to be confirmed that the methylation occurs before or at the same time as the processing. Technically, it is difficult to address this issue in vivo. Another approach can be used for this purpose is to make a point mutation in rDNA corresponding to this methylation site so that the methylation can not occur. It will be a very convincing evidence if a defect in the processing of the mutated 27S intermediate still occurs. However, methylation may be only temporally related to processing, but not required for processing. In yeast nop 1-3 mutant, the demethylated pre-rRNA was still processed (Tollervey et al., 1993). Yeast Dimlp, an 18S rRNA methylase, modifies a conserved site on 18S rRNA. Although the protein is essential, the methylation is not required for the processing (Lafontaine et al., 1995). Since there are 65 methylation sites on yeast rRNAs, it is possible that some of the methylations may be required for the processing of pre-rRNA and for the function of mature ribosome, while the others may be not essential. To determine if Nop2p might be directly involved in methylation, an in vitro methylation reaction was performed in this study. The possibility of direct catalysis of [methyl 3 H] transfer to an 27S in vitro transcript by Nop2p was investigated. The 27S transcript and Nop2p purified by immunoprecipitation were incubated in the buffer containing [ 3 H]-S-adenosyl-methionine (see Methods and Materials) at 30C for 1 hour. A 3.8-kb band corresponding to the 27S transcript was clearly visible on the gel stained with ethidium bromide. However, compared with the control reaction (without adding Nop2p), no isotope incorporation or any other change in the band pattern was seen (not shown). Since no positive control was included in this experiment, it is difficult to evaluate if the reaction conditions were appropriate for methylase activity. In the future, if available, a positive control, such as Dimlp (a yeast 18S methylase), should be included in the methylation reaction. The involvement of Nop2p in methylation could not be detected by the primer extension assay. This is most possibly due to the limitation of the method.

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126 However, it also suggests that there may be other possible mechanisms by which Nop2p functions in pre-rRNA processing. Nop2p and Protein Methylation Another possibility is that Nop2p may be a protein methylase. One piece of evidence to support this assumption is from the observation that a protein(s) seemed to be demethylated during Nop2p depletion, though the result is still inconclusive (Brokenbrough, J. S. and J. P. Aris, unpublished results). Based on the modified amino acid residues, protein methyltranferases can be classified into several groups. One group, protein methylase I, recognizes and modifies the arginine residues in the GAR domain (Lischwe, 1990; Paik and Kim, 1990). Nucleolin, fibrillarin and yeast Ssblp have been shown to be methylated on the arginine residues, though the function of, the enzymes responsible for, and the cellular location of, such modification have not been understood (Lischwe, 1990). Since there is no known RNA binding motif present in Nop2p sequence, and no association between Nop2p and RNA has been found, it is quite possible that Nop2p may not directly modify RNA. One scenario is that Nop2p may modify a protein with RNA methylase activity. The modified protein then transfers methyl groups to the UmGm v FUC2922 si te Or this site may be modified only after the cleavage at the C2 site in ITS2 has been completed. Nop2p may modify a protein component of a snoRNP complex, which is required for the processing at the C2 site. With such modification, the snoRNP may assume a functional conformation, bind to the 27S intermediate and cleave it at the C2 site. Since snoRNPs have been proposed to be involved in a variety of the events in ribosome synthesis, including modification and processing of rRNA, methylation of protein components in an snoRNP may be one of the mechanisms by which Nop2p functions in pre-rRNA processing.

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127 Nop2p and RNA Helicase ATP-dependent RNA helicases containing the conserved DEAD-box have been shown to be required for pre-rRNA processing, possibly by facilitating the formation of secondary structure of pre-rRNA. Drslp, Dbp3p, and Spb4p are all involved in the processing 27S intermediate to 25S rRNA (Sachs and Davis, 1990; Ripmaster et al., 1992; Weaver et al., 1997). A partial DEAD sequence is present in Nop2p (J. P. Aris, unpublished results). However, it is not known if the DEAD sequence in Nop2p plays any role in pre-rRNA processing. Since there is no evidence to support that Nop2p is associated with any RNA, it is unlikely that Nop2p will be directly involved in folding of pre-rRNA. Nop2p and SnoRNP Since Nop2p is involved in both rRNA methylation and the cleavage at C2 site in ITS2, it is possible that Nop2p may have multiple functions. One part of the protein with the SAM binding motif as the activity center may function as a rRNA or protein methylase. Another part may be involved in the cleavage. Nop2p is possibly not a nuclease, since no cleavage of a 27S rDNA transcript by Nop2p was observed in vitro, though the possibility can not be excluded that the transcript may not form the correct secondary structure. Nop2p may not be directly associated with any RNA. One possibility is that the part of the protein may be involved in association with a snoRNP through a weak protein-protein interaction. Such association may be required for the binding of the snoRNP to 27S intermediate, and therefore the nucleolytic activity of the snoRNP. Depletion of Nop2p made the protein less available for such association, resulting in a defect in cleavage at C2 site. In nop2-4 and nop2-5 strains but not in the other Ts strains, 27S intermediate was rapidly degraded at the restrictive condition, suggesting that the conformational change of

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128 the mutant protein may affect the binding of the snoRNP to 27S intermediate. The 27S intermediate without the binding of the snoRNP may become less stable and rapidly degraded. In conclusion, depletion and mutation of Nop2p indicate that Nop2p is required for synthesis of the 60S ribosomal subunit and processing of pre-rRNA to 25S and 5.8S rPvNAs. RNase protection suggests that Nop2p is involved in the methylation at the UmGm v FUC2922 site of 25S rRNA sequence. Sequence alignment reveals that Nop2p contains a SAM-binding domain. These results indicate that Nop2p may have methyltransferase activity. Since there is no RNA binding domain or motif present in the Nop2p sequence and since no RNA was found to be associated with Nop2p in immunoprecipitation assay, Nop2p may modify a protein component of a snoRNP complex. The modified snoRNP complex may then modify and process pre-rRNA. Through characterizing Nop2p function, this study helps to illustrate the molecular mechanisms by which nucleolar proteins function in modification and processing of prerRNA. Nop2p is an essential nucleolar protein with a significant sequence homology to human nucleolar protein pl20 (de Beus et al., 1994). Therefore, this study will also help reveal the role of human pi 20 protein in normal and tumor cells.

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BIOGRAPHICAL SKETCH Bo Hong is from Zhejiang Province in China. He attended medical school for five years and received his medical degree from Zhejiang Medical University in China in 1983. Two years later, he pursued research in basic medical science and received his Master of Medical Science degree from Shanghai Second Medical University in China in 1988. In the Fall of 1992, he joined the graduate program in cell and development biology at the University of Florida in the Department of Anatomy and Cell Biology. He completed his dissertation under the guidance of Dr. John P. Aris and received his Ph.D. degree in 1997. 137

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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 of Philosophy. f./L John P. Aris, Chairman Assistant 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 of Philosophy. William A. Dunn, Jr. r Associate 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 of Philosophy. AlfrefcT S. 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 Doctor of Philosophy. Maurice S. Swanson Associate Professor of Molecular Genetics and Microbiology

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This dissertation was submitted to the Graduate Faculty of the College of Medicine and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. August, 1997 fr wA'Av i. £ Dean, College of Medicine Dean, Graduate School

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UNIVERSITY OF FLORIDA 3 1262 08554 8203