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The Function of GW182 and GW/P Body in RNA Interference

Permanent Link: http://ufdc.ufl.edu/UFE0022438/00001

Material Information

Title: The Function of GW182 and GW/P Body in RNA Interference
Physical Description: 1 online resource (109 p.)
Language: english
Creator: Lian, Shangli
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: ago2, gw, gw182, mirna, rnai, sirna
Molecular Cell Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: RNA interference (RNAi) is an important mechanism that regulates gene expression and is conserved from plant to human. In this mechanism, small interfering RNAs (siRNAs) or microRNAs (miRNAs) specifically regulate the stability and translation of mRNA with complementary sequence in the encoding region or in the 3?-UTR. RNAi has become an important technique that is broadly adopted to study gene function in research laboratories and is also a new therapeutic strategy for a variety of diseases including viral infections, genetic diseases, and cancers. In our study, we discover that GW/P bodies (GWB), distinct cytoplasmic foci for mRNA degradation, are processing centers for mRNAs targeted by RNAi pathway. Knockdown of GW182, a GWB marker important for body formation, prevented the localization of Ago2, a key enzyme in RNAi pathway, to cytoplasmic foci and greatly impaired siRNA-mediated silencing. It suggested that GW182 and/or GWB were indispensable for RNAi process. Moreover, the number and size of GWB greatly increase when siRNA with a cellular target is introduced into mammalian cells. The increase of GWB correlated with siRNA-mediated silencing. Knockdown of GW182 or Ago2 greatly impaired RNAi function and abolished the siRNA-induced increase of GWB. Therefore, RNAi activity promotes the assembly of GWB which can potentially serve as cellular markers for monitoring RNAi activity during therapy. Furthermore, we reveal that GW182 is a possible suppressor in the miRNA-mediated silencing. GW182 interacts with human Ago1-4 proteins, the core components of RNAi silencing effecter complex. Interestingly, several non-overlapping regions of GW182 bind to the C-terminal half of Ago2 independently suggesting that GW182 may interact with multiple Ago proteins simultaneously. Moreover, the GW182-Ago interaction may recruit Ago2-miRNAs-mRNA complex to GWB for processing which contributes to the formation of GWB. Most importantly, tethering Ago2 or the C-terminal half of Ago2 to the 3?-UTR caused translational repression which required GW182. This implicated that GW182 was possibly the repressor brought to the 3?-UTR by Ago2 to switch off translation. Taken together, our study has advanced the understanding of the molecular and cell biology of RNAi and may potentially provide insight into future application and monitor of RNAi.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Shangli Lian.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Chan, Edward K.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022438:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022438/00001

Material Information

Title: The Function of GW182 and GW/P Body in RNA Interference
Physical Description: 1 online resource (109 p.)
Language: english
Creator: Lian, Shangli
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: ago2, gw, gw182, mirna, rnai, sirna
Molecular Cell Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: RNA interference (RNAi) is an important mechanism that regulates gene expression and is conserved from plant to human. In this mechanism, small interfering RNAs (siRNAs) or microRNAs (miRNAs) specifically regulate the stability and translation of mRNA with complementary sequence in the encoding region or in the 3?-UTR. RNAi has become an important technique that is broadly adopted to study gene function in research laboratories and is also a new therapeutic strategy for a variety of diseases including viral infections, genetic diseases, and cancers. In our study, we discover that GW/P bodies (GWB), distinct cytoplasmic foci for mRNA degradation, are processing centers for mRNAs targeted by RNAi pathway. Knockdown of GW182, a GWB marker important for body formation, prevented the localization of Ago2, a key enzyme in RNAi pathway, to cytoplasmic foci and greatly impaired siRNA-mediated silencing. It suggested that GW182 and/or GWB were indispensable for RNAi process. Moreover, the number and size of GWB greatly increase when siRNA with a cellular target is introduced into mammalian cells. The increase of GWB correlated with siRNA-mediated silencing. Knockdown of GW182 or Ago2 greatly impaired RNAi function and abolished the siRNA-induced increase of GWB. Therefore, RNAi activity promotes the assembly of GWB which can potentially serve as cellular markers for monitoring RNAi activity during therapy. Furthermore, we reveal that GW182 is a possible suppressor in the miRNA-mediated silencing. GW182 interacts with human Ago1-4 proteins, the core components of RNAi silencing effecter complex. Interestingly, several non-overlapping regions of GW182 bind to the C-terminal half of Ago2 independently suggesting that GW182 may interact with multiple Ago proteins simultaneously. Moreover, the GW182-Ago interaction may recruit Ago2-miRNAs-mRNA complex to GWB for processing which contributes to the formation of GWB. Most importantly, tethering Ago2 or the C-terminal half of Ago2 to the 3?-UTR caused translational repression which required GW182. This implicated that GW182 was possibly the repressor brought to the 3?-UTR by Ago2 to switch off translation. Taken together, our study has advanced the understanding of the molecular and cell biology of RNAi and may potentially provide insight into future application and monitor of RNAi.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Shangli Lian.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Chan, Edward K.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022438:00001


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THE FUNCTION OF GW182 AND GW/P BODY IN RNA INTERFERENCE By SHANG LI LIAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1

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2008 Shang Li Lian 2

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To my parents who have inspired and supported me to pursue my dream, to my mentor who has guided me through adversities and frustrations, to my husband who supports me every day 3

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ACKNOWLEDGMENTS It is with my greatest appr eciation and sincere thanks that I acknowledge the following individuals for their contributions to various aspects of my deve lopment as a scientist and as a person. Without their guidance, assistance a nd support, this work wouldnt be possible. First I would like to give my heartfelt tha nks to my mentor Dr. Edward Chan for his guidance through these years. I feel very fortunate to have chosen him as my mentor because to me he is the best mentor. He is always there for students. His wisdom, perseverance, patience, and support have guided me through th e adversities and frustrations in each step of the way. It was him who helps me develop fr om a very beginner to a person who is getting ready for the next step in the journey of science. I also feel very lucky to have ente red a very competitive area of research in the field of RNA interference, and have had the chance to get involved in the initial important studies from our laboratory. In addition, I am very grat eful to my committee: Dr. Maurice Swanson, Dr. Brian Harfe, Dr. Naohiro Tera da, Dr. Minoru Satoh, for their valuable time, effort, advice and support. Especially I need to thank Dr. Minoru Satoh for the help in statistical analysis and writing, and for the encouragement along my way. I would like to thank a ll of the members of the Chan la boratory for their technical and daily support, for the useful discussion and advice that make my work much more feasible, and for their friendship that make my graduate care er full of joyful and happy memories. Special thanks to John Hamel, a former coordinator of Chan laboratory, who taught me a lot of cloning techniques that benefit me a lot in my studies, and also to Andrew Jaky miw who is always ready to give valuable advice and help to many aspect s of my graduate career including experiments and writing. I am grateful and feel so lucky to have the chance working with Kaleb Pauley and Songqing Li. Their support, help, and fr iendship are a life-time treasure to me. 4

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I would like to extend my thanks to our co llaborator Dr. Marvin Fritzler and Dr. Theophany Eystathioy for providing human sera and assistance for experiments, and for reviewing my manuscripts. I need to thank Dr Naohiro Terada and Dr. Takashi Hamazaki for providing the EGFP3T3 cells. Special thanks to Dr. William McArthur who generously provides my NIH NIDCR training grant support. I am al so grateful to Dr.Witold Filipowicz, Dr. Jens Lykke-Andersen, Dr. Tom Hobman, Dr. Peter Saya ski, and Dr. Gordon Chan, for generously providing reagents. Finally, I would like to thank my parents for their unconditional love and support all these years. My father is my role model and his cr aving for knowledge and science has inspired me to take the journey of science. His advice and guidance in each step of my life have been invaluable. I am very lucky and grateful to have my sister being the best friend of mine who always supports and loves me. I need to tha nk my Aunt Lisa Lam a nd Uncle Kamchiu Lam who care about me and have been always there for me ever since I came to the United States. Lastly, I want to give special thanks to my husband for his love, understanding and support in each day and every step of the pursuit of my dreams. He has assisted me in a number of important experiments and been an integral part of my research career. 5

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF TABLES ...........................................................................................................................9LIST OF FIGURES .......................................................................................................................10ABSTRACT ...................................................................................................................... .............12 CHAPTER 1 INTRODUCTION ................................................................................................................ ..14GW182 and GW Body ............................................................................................................1 4GW/P Bodies Are Cytoplasmic Sites for Messenger RNA Decay .........................................14RNA Interference Is Linked to GW/P Body and GW182 ......................................................16RNA Interference ............................................................................................................16GW/P Body and GW182 Are Closely Correlated with siRNA and miRNA Silencing Function .......................................................................................................172 KNOCKDOWN OF GW182 DISRUPTS GW/P BODY AND IMPAIRS RNA INTERFERENCE .................................................................................................................. .19Introduction .................................................................................................................. ...........19Materials and Methods ...........................................................................................................20Antibodies .................................................................................................................... ....20Small Interfering RNA Synthesis ....................................................................................20Human Cell Culture and siRNA Transfection .................................................................20Fluorescence Microscopy ................................................................................................21Western Blotting .............................................................................................................. 22Statistical Analysis .......................................................................................................... 22Results .....................................................................................................................................23GW182 Is Essential for GWB Formation and Recruitment of Ago2 to GWB ...............23Silencing of Lamin A/C Was Im paired upon Knockdown of GW182 and Disassembly of GWB Using a Co-transfection Strategy .............................................23Silencing of Lamin A/C Was Impaired by Sequential Transfection of GW182siRNA and Lamin A/C-siRNA to HeLa Cells .............................................................24Discussion .................................................................................................................... ...........26Increased Number and Size of GWB upon si RNA Transfection May Correlate with mRNA Degradation Targeted by RISC .......................................................................26Integrity of GWB May Be Require d for Efficient RNAi Function .................................27 6

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3 SMALL INTERFERING RNA-MEDIA TED SILENCING INDUCES TARGETDEPENDENT ASSEMBLY OF GW/P BODIES ..................................................................35Introduction .................................................................................................................. ...........35Materials and Methods ...........................................................................................................37Antibodies .................................................................................................................... ....37Small Interfering RNA ....................................................................................................37Construction of Inducible GFP3T3 Fibroblast (TRE-GFP3T3) Cells ............................38Cell Culture and Transfection .........................................................................................39Western Blot Analysis .....................................................................................................40Fluorescence Microscopy ................................................................................................40Statistical Analysis .......................................................................................................... 41Results .....................................................................................................................................42The Size and Number of GWB Increased in Cells Transfected with siRNA Eliciting RNA Silencing of Its Endogenous Target ...................................................................42Small Interfering RNA Required Endogenous Expression of Its Target for Inducing an Increase in Size and Number of GWB ....................................................................43The siRNA-Induced Increase of GWB Star ted on Day 1 after Transfection and Lasted for at Least 4 Days ...........................................................................................44GW182 Was Required for the siR NA-Induced Increase of GWB ..................................45The siRNA-Induced Increase of GWB Re quired Ago2 and Correlated with RNA Silencing Activities ......................................................................................................46Knockdown of LSm1 or Rck/p54 Did not I nhibit the Assembly of GWB Induced by siRNA .....................................................................................................................47Discussion .................................................................................................................... ...........48Small Interfering RNA:mRNA Initiates Aassembly of Microscopic Detectable GWB by Recruiting GWB Components ......................................................................48The Role of GWB Components for the Assembly of GWB ...........................................49Regulation of GWB Assembly ........................................................................................514 THE C-TERMINAL HALF OF AGO2 BI NDS TO MULTIPLE GW-RICH REGIONS OF GW182 AND REQUIRES GW182 TO MEDIATE SILENCING ..................................69Introduction .................................................................................................................. ...........69Materials and Methods ...........................................................................................................70Construction of Deletion C onstructs of GW182 and Ago2 .............................................70Antibodies .................................................................................................................... ....73Plasmid Transfection, GST Pull-down, and Western Blot Analysis ...............................73Indirect Immunofluorescence ..........................................................................................74Tethering Assay Using a Dual Luciferase System ..........................................................74RNA Interference and Quantitative Real Time PCR ......................................................75Results .....................................................................................................................................75C-terminal Half of Huma n Ago2 Containing the PIWI Domain Was Responsible for the Interaction with GW182 ...................................................................................75GW182-Ago2 Interaction Wa s Important for the Lo calization of Ago2 in Cytoplasmic Foci .........................................................................................................77Ago2 Bound to Multiple Non-overla pping GW-rich Regions of GW182 ......................77 7

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The Interaction of Ago2 with GW182 Was Conserved in Other Human Ago Proteins ...................................................................................................................... ..78Tethering C-terminal Half of Ago2 to the 3-UTR of mRNA Recapitulated Ago2mediated Silencing Wh ich Required GW182 ..............................................................79Discussion .................................................................................................................... ...........80Formation of GW182 and Ago Protein Complexes ........................................................80C-terminal Half of Ago2 Preserved th e Silencing Function of Ago2 Probably Because It Maintained the Interaction with GW182 ....................................................815 DISCUSSION AND CONCLUSIONS ..................................................................................91GW/P Body Is a Processing Center for Me ssenger RNAs Targeted by RNAi Pathway with Its Component GW182 Playing a Criti cal Role in the Silencing Process ..................91Working Model and Conclusions ...........................................................................................92GW/P Body May Regulate Mu ltiple Cellular Processes ........................................................93LIST OF REFERENCES .............................................................................................................100BIOGRAPHICAL SKETCH .......................................................................................................109 8

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LIST OF TABLES Table page 5-1 Protein components of GW/P body ...................................................................................97 9

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LIST OF FIGURES Figure page 2-1 SMARTpool siRNA for GW182 cau sed the disassembly of GWB ..................................292-2 GW182-knockdown by siRNA completely disassembled GWB and abolished the localization of Ago2 and LSm4 to the foci ........................................................................302-3 The lamin A/C-knockdown by siRNA wa s impaired upon disassembly of GWB by co-transfecting lamin A/C-siRNA with GW182-siRNA ...................................................312-4 RNAi function was greatly affected by sequential transfecti on of GW182-siRNA and lamin A/C-siRNA ..............................................................................................................3 33-1 Transfection of siRNA for lamin A/C increased the size a nd number of GWB ................533-2 Transfection of siRNA for RAGE activated assembly of GWB .......................................553-3 Lamin A/C-siRNA did not induce stress granules .............................................................563-4 The siRNA-induced increase of GWB is target-dependent ...............................................573-5 Green fluorescence protein (GFP) wa s efficiently knocked down by siRNA ...................593-6 The increases of GWB started on day 1 and were most prominent on day 3 after transfection of siRNA for lamin A/C .................................................................................603-7 The increase of GWB induced by la min A/C-siRNA was prominent on day 3 ................623-8 GW182 was required for the siRNA-induced increase of GWB .......................................633-9 The siRNA-induced increase of GWB required Ago2 and correlated with RNA silencing activities ..............................................................................................................643-10 Knockdown of LSm1 or rck/p54 disassemble d GWB but did not inhibit the assembly of GWB induced by siRNA ...............................................................................................663-11 Lamin A/C-siRNA induced assembly of GWB and recruited re sidual rck/p54 to the reassembled GWB in rck/p54-knockdown cells ................................................................684-1 Identifying the interaction of C-termin al half of Ago2 with GW182 fragments using GST pull-down assays .......................................................................................................834-2 The GW182 fragment MGW formed insol uble complexes with Ago2 and C-terminal half of Ago2 .......................................................................................................................854-3 GW182 fragment MGW recruited Ago2 to cy toplasmic foci by interacting with the C-terminal half of Ago2 .....................................................................................................86 10

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4-4 The C-terminal half of Ago2 bound to mu ltiple non-overlapping GW-rich regions of GW182 ......................................................................................................................... ......874-5 Both GW182 fragments GW1 1 and MGW co-precipitated with other human Ago proteins ...................................................................................................................... .........884-6 Translational repression mediated by te thered C-terminal half of Ago2 required GW182 ......................................................................................................................... ......894-7 At least three non-overlapping GW-rich regions that are different from the orthologconserved GW-rich region can independently bind Ago2 .................................................905-1 A proposed model of the functi on of GW182 and GW/P body in RNAi ..........................985-2 Multiple cellular pathways associated with GWB .............................................................99 11

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE FUNCTION OF GW182 AND GW/P BODY IN RNA INTERFERENCE By Shang Li Lian August 2008 Chair: Edward K. L. Chan Major: Medical Sciences -Molecular Cell Biology RNA interference (RNAi) is an important mech anism that regulates gene expression and is conserved from plant to human. In this mech anism, small interfering RNAs (siRNAs) or microRNAs (miRNAs) specifically regulate the stability and translation of mRNA with complementary sequence in the encoding region or in the 3-UTR. RNAi has become an important technique that is broadly adopted to st udy gene function in research laboratories and is also a new therapeutic strategy for a variety of diseases including viral infections, genetic diseases, and cancers. In our study, we discover th at GW/P bodies (GWB), distinct cytoplasmic foci for mRNA degradation, are processing centers for mRNAs targeted by RNAi pathway. Knockdown of GW182, a GWB marker important for body formation, prevented the localization of Ago2, a key enzyme in RNAi pathway, to cy toplasmic foci and greatly impaired siRNAmediated silencing. It suggested that GW 182 and/or GWB were i ndispensable for RNAi process. Moreover, the number and size of GWB greatly increase when siRNA with a cellular target is introduced into mammalian cells. The increase of GWB correlated with siRNAmediated silencing. Knockdown of GW182 or Ago2 greatly impaired RNAi function and abolished the siRNA-induced increase of GWB. Therefore, RNAi activity promotes the 12

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13 assembly of GWB which can potentially serve as cellular markers for monitoring RNAi activity during therapy. Furthermore, we reveal that GW182 is a po ssible suppressor in the miRNAmediated silencing. GW182 interacts with human Ago1-4 proteins, the core components of RNAi silencing effecter complex. Interestin gly, several non-overlapping regions of GW182 bind to the C-terminal half of Ago2 independently suggesting that GW182 may interact with multiple Ago proteins simultaneously. Moreover, the GW182-Ago interac tion may recruit Ago2miRNAs-mRNA complex to GWB for processing whic h contributes to the formation of GWB. Most importantly, tethering Ago2 or the C-te rminal half of Ago2 to the 3-UTR caused translational repression which required GW182. This implicated that GW182 was possibly the repressor brought to the 3-UTR by Ago2 to switch off translation. Taken together, our study has advanced the understanding of the molecular and cell biology of RNAi and may potentially provide insight into future a pplication and monitor of RNAi.

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CHAPTER 1 INTRODUCTION GW182 and GW Body In 2002, GW182 is identified and cloned by our laboratory as a novel autoantigen using autoimmune serum from a patient with motor and sensory neuropathy. GW182 is a 182 kDa protein with a classical RNA recognition motif at its C-terminus and is characterized by multiple glycine/tryptophan (GW) repeats. GW182 is found to be associated with a specific subset of mRNAs and consistently reside within unique cyt oplasmic foci designated as GW bodies that are distinct from other known cytoplasmic organell es such as Golgi complex, endosomes, lysosomes or peroxisomes (Eystathioy et al. 2002a). It is speculated that GW bodies are involved in the post-transcriptional regulation of gene expression by sequestering a subset of gene transcripts involved in cell growth and homeost asis. GW bodies are small, ge nerally spherical, cytoplasmic foci that vary in number and size at di fferent stages of the cell cycle (Yang et al. 2004). Electron microscopy demonstrates that GW bodie s are electron dense st ructures of 100 nm in diameter devoid of a lipid bilayer membrane. These structures co mprise of clusters of electron dense strands of 8 nm in diameter. In vi tro gene knockdown of GW 182 using short hairpin RNA (shRNA) plasmid results in instability and disappear ance of GW bodies (Yang et al. 2004). Autoantibodies to GW182/GW bodies are typically found in patients with Sjgrens syndrome, mixed motor/sensory neuropathy, and systemic lupus erythematosus (Eystathioy et al. 2003a;Bhanji et al. 2007). GW/P Bodies Are Cytoplasmic Sites for Messenger RNA Decay In the past decade, studies have shown that the 5-3 mRNA degradation factors, including Xrn1 (5-3exonuclease), Dcp2:Dcp1 (decapping enzy me), and LSm1-7 complex (stimulator of mRNA decapping), colocalize in distinct cytoplasmic foci (Heyer et al. 1995;Bashkirov et al. 14

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1997;van Dijk et al. 2002;Ingelfinger et al. 2002). However, the function of these cytoplasmic foci is unclear. In 2003, Sheth and Parker report that in yeast these cy toplasmic foci contain mRNA degradation intermediates in addition to the 5-3 mRNA decay factors and designate the foci as processing bodies (P bodies) (Sheth and Parker, 2003). They propose that P bodies are dynamic sites involved in the regulation of mR NA degradation and storage, and that the flux of mRNAs between polysomes and P bodies are a critical aspect of cytoplasmic mRNAs metabolism. In mammalian cells, decay factor s Dcp1 and LSm4 co-localize with GW182 in GWB, which is shown to contain poly (A)+ RNA and dynamically disappear as mRNA breakdown was abolished (Eystathioy et al. 2003b;Cougot et al. 2004). Therefore, GWB are considered as the mammalian analogues of P bodies and as the sites for active 5-3 mRNA degradation, which are designated here provisionally as GW/P bodies (GWB). In addition to the 5-3 mRNA decay pathway, GWB are also considered sites for the nonsense-mediated decay (NMD) and AU-rich el ement (ARE)-mediated decay pathways. NMD is an mRNA quality control mechanism that degrades aberrant mRNAs having a premature translational termination codon (PTC), there by preventing the synthesis of truncated and potentially harmful proteins (Conti and Izaurralde, 2005). Dcp1:Dcp2 decapping complex is shown to be associated with Upf1, a component central to NMD (Muhlrad and Parker, 1994;He and Jacobson, 1995;Lykke-Andersen, 2002), and deca pped mRNAs in NMD. Depletion of the decapping complex subunit Dcp2 results in impaired NMD (Lejeune et al. 2003). Inhibition of NMD reveals that mRNA and NMD factors are dynamically and sequentially recruited to GWB (Durand et al. 2007). AU-rich elements (AREs) are found in the 3 untranslated region (3UTR) of a variety of short-lived mRNAs in mammalian cells. The human Dcp1:Dcp2 complex and other mRNA decay enzymes recruit ARE-cont aining mRNAs via Tristetraprolin (TTP), the 15

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ARE-binding protein that activates mRNA d ecay (Lykke-Andersen and Wagner, 2005). TTP interacts with Dcp2 and decapping activator Edc3 to activate decapping (Fenger-Gron et al. 2005;Franks and Lykke-Andersen, 2007). In addition, TTP and its paralog BRF-1 could nucleate GWB formation to silence AREmRNAs (Franks and Lykke-Andersen, 2007). RNA Interference Is Linked to GW/P Body and GW182 RNA Interference RNA interference (RNAi) is initially describe d in plants as a genetic control mechanism implicated in virus resistance (Ratcliff et al. 1997;Covey et al. 1997), genome maintenance (Assaad et al. 1993) and developmental control (Boerjan et al. 1994). This mechanism is further characterized in C. elegans. as a potent a nd sequence specific mech anism that silences endogenous genes (Fire et al. 1998). Based on up-to-date stud ies, RNAi includes siRNAand miRNA-mediated silencing. In siRNA-mediated silencing, the dsRNAs, which are formed in cells or are introduced into cells by viral infect ion or artificial expr ession, are processed by RNase III enzyme, Dicer, into ~20-bp double-stranded small interfering RNAs (siRNAs). The siRNAs are then unwound and the antisense st rands are incorporated into RNA-induced silencing complex (RISC). Subsequently, RISC binds to and cleaves target mRNA with complete complementary sequence to the siRNA (Filipowicz et al. 2005). Argonaute 2 (Ago2) is the core component of RISC and harbors RNase H activity responsible for the cleavage of target mRNA (Liu et al. 2004). MicroRNAs (miRNAs) are endogenous ~21-nt re gulatory RNAs that are evolutionarily conserved in most species and are estimated to regulate ~30% of protein-encoding genes in human (Lewis et al. 2005). MiRNAs are processed from endogenous precursor molecules folded into hairpin-like structure. The matura tion of miRNAs includes two steps, both catalyzed by enzymes of the RNase III family, Drosha and Di cer. Drosha is responsible for the processing 16

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of primary miRNA transcripts (pri-miRNAs) to ~70-nt hairpins named precursor miRNAs (premiRNAs). Subsequently, Dicer processes pre-mi RNAs into mature miRNAs, which binds to the 3-UTR of target mRNA with imperfect complementary sequence and regulates gene expression by increasing instability or repressing translation of target mRNA (Filipowicz et al. 2005). Like siRNA, miRNA also forms RISC-like ribonucleopro tein particles (miRNPs). Argonaute proteins are the core components of both RISC and miRNPs. GW/P Body and GW182 Are Closely Correl ated with siRNA and miRNA Silencing Function As a potentially powerful tool for experimental gene knockdown and clinical therapy, RNAi has been extensively studied over the past decade. However, many of the studies on RNAi have only used biochemical techniques with little attention to th e cell biology of RNAi. Recently evidence have linked mRNA turnover to RNAi at the level of cell biology. Exonucleases AtXrn4 (A. thaliana) and dXrn1 (D. melanogaster), whose mammalian orthologue Xrn1 is a GWB component (Cougot et al. 2004), are demonstrated to be required for degradation of the 3 fragme nt of RISC-targeted mRNA. Knockout of AtXrn4 or dXrn1knockdown by siRNAs results in accumulation of the 3 fragment of the cleaved mRNA (Gazzani et al. 2004;Souret et al. 2004;Orban and Izaurralde, 2005). Interestingly, further studies show that all human A go proteins localize to GWB and that Ago2 interacts with GWB components Dcp2 and Xrn1 (Sen and Blau, 2005;Pillai et al. 2005;Liu et al. 2005b). In addition, miRNAs and their target mRNAs are pres ent in GWB and the targ et mRNAs localize to the foci in a miRNA-dependent manner (Pillai et al. 2005;Liu et al. 2005b). We are one of the first groups to describe the link between RNAi and GWB/GW182. Two studies from our laboratory strongly support this correlation. The first study focusing on siRNAmediated function shows that tr ansfected Cy3-labeled siRNAs ar e present in GWB and could be 17

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18 immunoprecipitated by anti-GWB serum. More importantly, disruption of GWB by dominant interfering mutants significantly impa irs siRNA silencing function (Jakymiw et al. 2005). The other study focusing on miRNA-mediated function s hows that, similarly, transfected Cy3-labeled let-7 miRNAs localize to GWB and could be det ected in the immunoprecipitate using an antiGWB serum. Moreover, knockdown of Drosha or DGCR8, the two factors forming the microprocessor complex to mediate the biogenesi s of miRNAs, leads to a blockage in the maturation of miRNAs and disassembly of GW B. Transfecting siRNA into the Droshaknockdown cells reassembles GWB (Pauley et al. 2006). In addition to siRNA/miRNA, we demonstrate that the key fact or of RNAi, Ago2, interacts wi th GW182 suggesting that GW182 could be important for siRNAand miRNA-mediated function in human cells. Studies of GW182 orthologs in Drosophila and C. elegan s strongly support this Knockdown of the Drosophila GW182 ortholog greatly impairs miR NA-mediated gene silencing activity in Drosphila cells (Rehwinkel et al. 2005). The C. elegans GW182 ortholog, AIN-1, is capable of interacting with an Ago protein and Dicer, and may target the A go protein to cytoplasmic GW/P bodies in C. elegans (Ding et al. 2005). With all these evidence, we further characterize the function of GW182 and GWB in RNAi in the current study. We propose a model in which GWB are cytoplasmic processing centers for the mRNAs targeted by RNAi pathway with GW182 playing an important role in the pathway.

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CHAPTER 2 KNOCKDOWN OF GW182 DISRUPTS GW/P BODY AND IMPAIRS RNA INTERFERENCE Introduction RNA interference (RNAi) is an efficient m echanism for post-transcriptional, sequencespecific regulation of gene expression and is co nserved from plants to mammalian cells. Much research has been performed to gain a better und erstanding of RNAi from a biochemical point of view in the past few years, but little is known about intracellular location of RNAi activity. Only recently has the evidence been reported linki ng RNAi to GW/P bodies (GWB). One of the evidence is that exonuclease AtXrn4 ( A. thaliana ) or dXrn1 (D. melanogaster ) are required for degradation of 3 fragment of RISC-targeted mRNA (Gazzani et al. 2004;Souret et al. 2004;Orban and Izaurralde, 2005). In addition, Ago2, a key factor in RNAi, localizes to GWB and interacts with GW182 in mammalian cells (Sen and Blau, 2005;Jakymiw et al. 2005;Liu et al. 2005b). The localization of A go2 is not altered by the presen ce or absence of siRNAs or their target mRNAs (Sen and Blau, 2005). Moreover, siRNAs, miRNAs and the mRNA targets are presence in GWB (Pillai et al. 2005;Jakymiw et al. 2005;Liu et al. 2005b). All these intriguing observations indicate that certain stage along RNAi or miRNA pathway occurs in GWB and prompt us to ask the following quest ions: (a) How GWB participate in RNAi? (b) What is the effect of disruption of GWB on RN Ai activity? Studies fr om our laboratory has demonstrated that expression of the N-terminal 1/3 fragment of GW182 or the C-terminal 1/2 fragment of Ago2 is able to disrupt endoge nous GWB formation and impair the silencing capability of siRNA to lamin A/C (Jakymiw et al. 2005). These results suggest that the GW182 protein and/or the microenvironment of GWB ma y contribute to the RISC and RNAi activity. However, the mechanisms on how these constructs disrupt GWB remain puzzling, which create uncertainty of the effect of GWB on RNAi. In this study, to further prove the importance of 19

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GWB and GW182 for RNAi function, siRNA for GW 182 is employed as a different method to disrupt GWB. The effect of siRNA for GW182 on RNAi activity is measured by the expression of endogenous lamin A/C. Materials and Methods Antibodies The human prototype anti-GW182 and anti-hAgo2 sera were obtained from serum banks at the Advanced Diagnostics Laboratory, University of Calgary and the Division of Rheumatology and Clinical Immunology, University of Flor ida. Prototype human anti-GW182 serum was described previously (Yang et al. 2004). Rabbit anti-LSm4 pol yclonal antibody was produced as described (Eystathioy et al. 2002b). Mouse monoclonal antilamin A/C 636 and anti-tubulin were purchased from Santa Cruz Biotec hnology, Inc. and Sigma-Aldrich, respectively. Small Interfering RNA Synthesis The lamin A/C-siRNA targeting region was sele cted from a 21-nt target mRNA (position 608 relative to the start codon). The Cy3-5-e nd labeled antisense an d sense lamin siRNA duplex (5Cy3-UGU UCU UCU GGA AGU CCA GdTdT3 and 5P-CUG GAC UUC CAG AAG AAC AdTdT3, respectively) was chemi cally synthesized by Dharmacon. The Cy3labeled Luciferase GL2 siRNA duplex and siGENOME SMARTpool reagent human TNRC6 (GW182) siRNA duplex were purch ased from Dharmacon. The aforementioned duplexes were resuspended in 1x siRNA Univer sal buffer and the resulting 20 M stock was stored in aliquots at -20oC prior to use. Human Cell Culture and siRNA Transfection HeLa cells were cultured in Dulbeccos Modification of Eagles Medium (DMEM) containing 10% fetal bovine serum (FBS) in a 37oC incubator with 5% CO2. HeLa cells were grown on coverslips to 30-40% confluency in a 6-well plate. The following day the media was 20

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replaced with 1 ml of Opti-MEM I Reduced Serum Medium (Invitroge n) containing 10% FBS before transfection. In single siRNA transfection, 100 nM or 200 nM siRNAs were transiently transfected into HeLa cells using 7L of Oligofectamine (Invitrog en). In siRNA co-transfection, 100 nM siRNA for GW182 were mixed with 100 nM siRNA for lamin A/C and were cotransfected into HeLa cells using 7L of Oligofectamine. In siRNA sequential transfection, 200 nM or 40 nM siRNA for either GW182 or luciferase were transfected into cells using 7L of Oligofectamine. 24 hours after the 1st transfection, the media was re placed with 1 ml of OptiMEM I Reduced Serum Medium (Invitrog en) containing 10% FBS. In the 2nd transfection, either 200 nM siRNA for lamin, or 20 nM siRNA for GW182 were mixed with 40 nM siRNA for lamin A/C and then were transfected into HeLa cells using 7L of Oligofectamine. In each single transfection mix was added to the well and the cells were incubated at 37oC in a CO2 incubator for 24 hrs, after which the media wa s replaced with normal growth media. After certain hours of growth the cells were fixed and processed for indire ct immunofluorescent studies. Fluorescence Microscopy Indirect immunofluorescence analyses used adherent HeLa cells grown on glass coverslips in 6-well culture dishes. All transfected cells were rinsed fi rst with phosphate buffered saline (PBS), fixed in 3% paraformaldehyde at room temperature for 10 minutes and permeabilized with 0.5% Triton X-100 at room temperature for 5 minutes. For colocalization studies, cells were incubated at room temperature for 1 hour wi th primary antibodies to the following proteins: GW182 (human serum, 1:6000), Ago2 (human se rum, 1:200), lamin A/C (mouse monoclonal antibody, 1:100), LSm4 (rabbit polyclonal antibody, 1:200) After washing with PBS, cells were incubated with corresponding secondary fluor ochrome-conjugated goat antibodies at room temperature for 1 hour. Alexa Fluor 488 (1:400), Alexa Fluor 568 (1:400) (Invitrogen) and Cy5 21

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(1:100) (Jackson ImmunoResearch Laboratories) were the fluorochromes used. Lastly, the coverslips were mounted onto glass slides using VECTASHIELD Hard Set Mounting Medium with 4,6-diamidino-2-phenylindole (DAPI, VECTOR Laboratories) Fluorescence images were obtained using a Zeiss Axiovert 200M microscope. Images of fixed cells were taken using 20x 0.75 NA or 40x 1.4 NA objectives. Western Blotting HeLa cells were grown on a 6-well plate without coverslip. In each experiment, transfected cells were harvested in RI PA buffer (150 mM NaCl, 1% TX-100, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH7.5) containing Complete protease cocktail inhibitors (Roche). Afterward the protein co ncentration of each sample was measured, equal amounts of protein extract were separated on a 10% polyacrylamide gel and transferred to nitrocellulose. The nitrocellulose membrane was blocked in 5% nonfat dried milk in PBSTween 1 hour at room temperature, then probed with primary antibody in appropriate dilution for 1 hour, followed by incubation with horseradish peroxidase-conjuga ted goat anti-mouse or antihuman IgG (1:5000, Caltag) for 1 hour at room temperature. Immunoreactive bands were detected by the SuperSignal Chemiluminescent sy stem (Pierce) according to the manufacturers instructions. Statistical Analysis The expression of lamin A/C in each cell was monitored individually based on the light intensity using the AxioVs40 software (Ver. 4.4. 0.0, Carl Zeiss Vision GmbH). Images from a complete experiment were taken using the sa me exposure time and at least 100 cells were randomly selected for the measurement of lami n A/C intensity. Results were analyzed for statistical significance using Prism 4.0c for M acintosh (Graphpad Software Inc., San Diego, 22

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CA). The median values of lamin A/C signal in untransfected HeLa cells and lamin A/C-siRNA transfected HeLa cells were defined as 0 and 100% siRNA function respectively. Results GW182 Is Essential for GWB Formation and Recruitment of Ago2 to GWB Although the integrity of GWB was shown important for effici ent silencing activity, issue is raised against the unknown mechanism on how the dominant interfering constr ucts disrupted GWB (Jakymiw et al. 2005). One may argue that it was the constructs themselves rather than disruption of GWB that interfered with the RISC activ ities. A different method to disassemble GWB is necessary to confirm the important ro le of GWB on RNAi. Here we employ a siRNA pool which contains 4 different individual siRNA targeting di fferent sites along GW182 mRNA, to silence the expression of GW182 and lead to disassembly of GWB. To test the efficiency of siRNA pool silencing target GW182, HeLa cells were transfected with 200 nM of siRNA pool for 48, 72 and 96 hours and changes of GWB were monitored by staining with human antiGW182 antibody. As shown, the majority of GW B were disassembled upon transfection of the siRNA pool for GW182 after 48 hours (Fig. 2-1). More complete disruption of GWB was achieved after 72 and 96 hours (Fi g. 2-1). Furthermore, the localization of Ago2 and LSm4 to these cytoplasmic foci was completely a bolished upon GW182-knockdown (Fig. 2-2). In summary, our data support that GW182 is essent ial for GWB formation and localization of Ago2 to GWB. Silencing of Lamin A/C Was Impaired upo n Knockdown of GW182 and Disassembly of GWB Using a Co-transfection Strategy To test our hypothesis that GW182 and/or GWB are importa nt for RNAi function, HeLa cells were transfected with siRNA specific for GW182, siRNA for lamin A/C, or both for 3 days, and double-stained with anti-GW182 and anti-l amin A/C antibody (Fig. 2-3A). GW182-siRNA 23

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caused disassembly of GWB without significantly affecting the expression of lamin A/C. In contrast, both number and size of GWB increas ed in siRNA transfected cells where the expression of lamin A/C was effi ciently silenced by siRNA specifi c for lamin A/C (Fig. 2-3A). Notably, co-transfection of si RNA specific for GW182 and siR NA specific for lamin A/C did not result in larger and greater number of GWB formation. Instead, it led to complete disassembly of GWB and the efficiency of lamin A/C-knockdown by siRNA was dramatically impaired (Fig. 2-3A). Western blot analysis of lamin A/C expressi on also demonstrated knockdown of lamin A/C was impaired in cells co-transfected with siRNA for GW182 and siRNA for lamin A/C, compared to that in cells transfected with siRNA for lamin A/C alone (Fig. 2-3B). Altogether, it is impli cated that knockdown of GW182 abolishes the recruitment ability of siRNA and prevents formation of cytoplasmic foci which leads to inhibition of efficient RNAi activity. A potential issue of co-transfection of siRNA for GW 182 and siRNA for lamin A/C is that the siRNA for GW182 may compete with th at for lamin A/C on the intracellular RNAi machinery resulting in incomplete knockdown of either GW182 or lamin A/C. Although there was no evidence that the knockdown of GW182 or di sruption of GWB was less complete in the cells transfected with mixed siRNA compared to those transfected with siRNA for lamin A/C alone, it is possible that the reduction of lami n A/C silencing in the mixing siRNA transfection may not be directly related to GWB disassembly but was a result from competitive usage of RNAi machinery. Silencing of Lamin A/C Was Impaired by Sequential Transfection of GW182-siRNA and Lamin A/C-siRNA to HeLa Cells To further confirm that GW182 and/or GWB are important for RNAi, we employed an alternative strategy using sequential transfection. HeLa cells were first transfected with either 200 nM siRNA targeting GW182 or 200 nM siRNA fo r luciferase and then two days later the 24

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cells were transfected with 200 nM siRNA specific to lamin A/C (Fig. 2-4A). Consistent with the co-transfection data above, GW182-knockdown significantly impaired RNAi activity while transfection of luciferase siR NA did not (Fig. 2-4A). Quantif ication of lamin A/C expression level in 100 cells for each sett ing demonstrated a 91% reduc tion of RNAi activity in GW182knockdown cells (Fig. 2-4B), which was statistica lly significant from that in control cells transfected with siRNA for luciferase (Fi g. 2-4B, P<0.001, Kruskal-Wallis test) but was not significantly different from that in untransfected cells. In c ontrast, the reduction of RNAi function was only 16% in cells transfected with luciferase siRNA which was not statistically significant compared to that in cells with la min A/C-siRNA alone (P>0.05). An alternative consideration was that a substantially lower conc entration of siRNA could be employed for these analyses because the siRNA titration experiment showed that as low as 40 nM siRNA were efficient in ~95% knockdown 48 hours post-transfec tion (data not shown). Thus experiments 2 and 3 as shown were performed with 40 nM of siRNA for GW182 or siRNA for luciferase in the first transfection, which was followed by a second transfection of 40 nM siRNA for lamin A/C 48 hours later (Fig. 2-4B). Additional 20 nM siRNA for GW182 was included in the second transfection with an atte mpt to maintain a lower level of GW182. In control cells, 20 nM siRNA for luciferase was also added in the second tran sfection (Fig. 2-4B). No tably, reduction of RNAi function in cells transfected with 200 nM of siRNA for GW18 2 (experiment 1) was more dramatic than that in the cells with 40 nM of siRNA (experiment 2 a nd 3). Immunofluorescence data were consistent with this result showi ng that disruption of GWB was more complete by using 200 nM of siRNA for GW182 than 40 nM of siRNA upon 48-hour transfection (data not shown), indicating reduction of RN Ai activity may correlate with the degree of disassembly of 25

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GWB. Again, our data demonstrates that GW 182 and formation of GWB are important for efficient RNAi activity. Discussion In the present study, knockdown of GW182 by siRNA using both co-transfection and sequential transfection strategy resu lted in disruption of GWB, abo lishment of the localization of Ago2 to GWB, and inhibition in siRNA-mediated si lencing. Our data is consistent with our recent observation that disruption of GWB using independent dominant negative constructs of GW182 or Ago2 led to impaired si RNA silencing activity (Jakymiw et al. 2005). The implication that RNA silencing is localized to these cytoplasmic foci is intriguing and open interesting potentials for improving RNAi in expe rimental manipulation a nd potential therapeutic applications. Increased Number and Size of GWB upon siRNA Transfection May Correlate with mRNA Degradation Targeted by RISC In this report, we observed that transfecti ng lamin A/C-siRNA induced larger and greater numbers of GWB where GW182 and Ago2 localize. The number and size of GWB reached a peak on the 3rd day post-transfection (data not sh own). The fact that detectable changes of GWB take place at 24 hours after the initial silencing activity of the transfected siRNA suggests the changes are related to mRNA degradation targeted by RISC, which is consistent with the proposed function of GWB in mRNA decay. Moreover, the number and size of GWB may correlate with the amount of mRNA need to be de graded within the cytoplasm. It is supported by that cells transfected with siRNA for luciferase, a protein doesnt exist in human cells, did not have increased GWB. This may be due to no RISC-targeted mRNA sitting on GWB and no recruitment of decay factors for mRNA degradati on. Nevertheless, it is possible that GWB may also involve in helping RISC to target and cleave mRNA at ear lier stage of RNAi activity. 26

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Chances are earlier changes of GWB were too mi nor to detect. Overall, our results suggest siRNA is capable of localizing target mRNA to GWB and forming larger foci to participate potentially in silencing activity. This model is supported by recent studies which indicate that miRNA is very important for recruiting the targeted mRNA to GWB (Liu et al. 2005b). Integrity of GWB May Be Required for Efficient RNAi Function In our laboratory, three diffe rent strategies have been used to disassemble GWB and examine the effect of GWB on RN Ai: 1.Dominant negative effect us ing two different constructs (the C-terminal half of Ago2 and the N-terminal 1/3 of GW182) (Jakymiw et al. 2005); 2. Cotransfection strategy in which siR NA for GW182 and lamin A/C were co-transfected into cells; 3. Sequential strategy in which siRNA for GW182 wa s transfected into cel ls prior to a second transfection of siRNA for lamin A/C. All thes e strategies have consistently demonstrated impaired RNAi function upon GWB disassembly. In contrast, cells with intact GWB showed efficient silencing activity of siRNA on its target The limitation of present study is that RNAi mechanism was used to silence GW182 which is obvious very important for RNAi silencing process, leading to self-limiting compro mised knockdown of GW182. This incomplete knockdown of GW182 may conceal the impor tance of GW182 on RNAi, although GW182knockdown by 200 nM siRNA in our study already greatly affected the siRNA mediatedsilencing. From the results altogether, we propose th at GW182 help to fo rm GWB by recruiting Ago2/RISC, siRNA/miRNA, targeted mRNA, mRNA d ecay factors, and other proteins that are required for RNAi or mRNA decay. GWB provid e a platform or microenvironment where Ago2, siRNA/miRNA, and targeted mRNA can be positio ned properly and where targeted mRNAs are cleaved by RISC with high efficiency. Although evidence has been shown that purified Ago2 with siRNA can manage to cleave the targeted mRNA in vitro, the localization of RISC to GWB 27

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may greatly increase the cleavage efficiency in vivo It is supported by th e data that abolishing localization of Argonaut 2/RISC to GWB by e ither overexpression of dominant-negative constructs or GW182-knockdown gr eatly inhibited RISC activit y. Moreover, GWB may also help to recycle siRNAs there by increasing the knockdow n efficiency of the target. However, further studies will be needed to elucidate these mechanisms. *This work was published in Nature Cell Biology, 2005 Dec, Vol 7 (12):1267, and Cell Cycle, 2006 Feb, Vol 5 (3):242. 28

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Figure 2-1. SMARTpool siRNA fo r GW182 caused the disassembly of GWB. HeLa cells transfected with siRNA for GW182 for 48, 72, and 96 hours, were stained with index human anti-GW182 antibody (green). Nuclei we re counterstained with DAPI (blue). Compared with untransfected cells in panel i showing many cells with GWB (arrows), majority of GWB were disassemb led in cells transfected with siRNA for GW182 after 48 h (ii). More complete di sruption of GWB was achieved after 72 h (iii) and 96 h (iv). Few cel ls (u) with apparently intact GWB were observed representing potentially unt ransfected cells. Bar, 10 m. 29

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Figure 2-2. GW182-knockdown by si RNA completely disassembled GWB and abolished the localization of Ago2 and LSm4 to the foci. HeLa cells we re transfected with 200 nM pooled siRNA for GW182 for 72 hours, stained with index human anti-Ago2 serum (green, ii) and rabbit anti-LSm4 (red, iv). Nuclei were counterstained with DAPI (blue). Compared with untransfected cells stained with anti-Ago2 (i) and anti-LSm4 (iii) showing many cells with cytoplasmic foci that Ago2 and LSm4 enriched in (arrows), the localization of Ago2 and LSm4 to these cytoplasmic foci was completely abolished in cells transfected with siRNA specific for GW182 after 72 h (ii, iv). In addition to lo calization to GWB, LSm4 is also known to be involved in RNA splicing function in the nucleus (iii iv) which is not affected by the GW182siRNA transfection. Merge images are shown in the bottom row. Bar, 10 m 30

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Figure 2-3. The lamin A/C-knockdown by siRNA was impaired upon disassembly of GWB by co-transfecting lamin A/C-siRNA with GW182-siRNA. A) HeLa cells were transfected with 100 nM siRNA for GW182 ( iii, iv), 100 nM siRNA for lamin A/C (v, vi), or both (vii, viii) for 3 days, and were processed for double IIF with human antiGW182 antibody (green) and mouse monoclona l antibody to lamin A/C (magenta). Nuclei were counter stained with DAPI (blue). GW 182-siRNA caused disassembly of GWB (iii) as expected without affecting the expression of lamin A/C (iv, compared to ii). In contrast, the transf ection of siRNA for lamin A/ C showed both increased in number and size of GWB (v, arrows) and e fficient silencing of lamin A/C (vi). Notably, co-transfection of siRNA for GW182 and lamin A/C resulted in almost complete disassembly of GWB (vii, compared to i and iii) and dramatically impaired 31

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lamin A/C expression (viii, compared to vi). Arrows, GWB. Bar, 10 m. B) Western blot analysis of lamin A/C expression demonstrating inhibition of siRNA silencing activity by co-tra nsfection of siRNA specific for GW182. Tubulin levels were monitored to confirm the equal loading of samples. 32

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Figure 2-4. RNAi function was gr eatly affected by sequential transfection of GW182-siRNA and lamin A/C-siRNA. A) Transfection of siRNA for GW182 disassembled GWB and significantly inhibited RNAi. Cells were co-stained with index human anti-GW182 antibody (green) and mouse monoclonal anti-lamin A/C antibody (magenta). Nuclei were counterstained with DAPI (blue). Th e expression of lamin A/C was efficiently silenced either in cells singly transfected w ith 200 nM siRNA to lamin A/C (iv) or in sequential transfection experiments where 200 nM control siRNA for luciferase was transfected prior to the addi tion of 200 nM siRNA for lamin A/C (viii). Note that both conditions obviously increased the size a nd number of GWB to different degrees (iii, vii, compared to i and v). In cont rast, cells first transfected with siRNA for GW182 showed disassembly of GWB (v) and im paired silencing of lamin A/C in the 2nd transfection by siRNA for lamin A/C (vi) Arrowheads show examples of cells 33

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34 with disrupted GWB and failure to silence la min A/C. Some cells (*) with significant silencing of lamin A/C have a few detectable GWB (v, arrows). Arrow, GWB. Bar, 10 m. B) Quantification of the effect of GW182 knockdown and GWB disassembly on siRNA functional activity. Results of three experiments using different concentrations of siRNA are presented. Th e lamin A/C intensities for at least 100 cell nuclei were measured for each data point and the value of untransfected and lamin A/C-siRNA transfected cell nuc lei were established as 0 and 100% siRNA function. The percent siRNA function was calculate d based on median value of lamin A/C intensity in each group. Cells transfect ed with siRNA for GW182 had significant decrease in RNA silencing function (9 %) compared to those transfected with control luciferase (luc) siRNA (P<0.001, Kruskal-Wallis test). In contrast, cells transfected with siRNA for luciferase yi elded between 76% to 93% siRNA function and were not statistically different from control with transfection of lamin A/CsiRNA alone (P>0.05, **). In experiment 1 using highest con centration of siRNA (200 nM) for each transfection, intensity of lamin A/C in GW182-siRNA transfected group did not show statistically differen ce with that of the untransfected group (P>0.05, *).

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CHAPTER 3 SMALL INTERFERING RNA-MEDIATED SI LENCING INDUCES TARGET-DEPENDENT ASSEMBLY OF GW/P BODIES Introduction GW bodies (GWB), also known as mammalia n processing bodies (P bodies), are cytoplasmic foci that contain multiple decay factors and are involved in the 5 3 mRNA degradation pathway. GWB are named from the marker protein GW182, which contains multiple glycine (G) and tryptophan (W) repeat s and a classic RNA binding domain at the carboxyl terminal (Eystathioy et al. 2002a). The mRNA decay factors/complexes found in GWB include the deadenylase Ccr4, the decapping complexes Dcp1a/1b/Dcp2, the LSm1-7 complex, Ge-1 (also known as Hedls) rck/p54, and exonuclease Xrn1 (Bashkirov et al. 1997;van Dijk et al. 2002;Ingelfinger et al. 2002;Lykke-Andersen, 2002;Eystathioy et al. 2003b;Cougot et al. 2004;Andrei et al. 2005;Yu et al. 2005;Fenger-Gron et al. 2005). GWB are physically juxtaposed to and transiently inte ract with stress granules (SG), which process cytoplasmic aggregates of stalled translationa l preinitiation complexes that accumulate during stress responses and share certain components with GWB (Kedersha et al. 2005). In addition to mRNA decay, a crucial ro le of GWB and their components in RNA interference (RNAi) was recently uncovered (Anderson and Kedersha, 2006;Jakymiw et al. 2007;Eulalio et al. 2007a). RNAi is a posttranscriptional gene silencing mechanism that uses specific double-stranded RNA to silence genes in a sequence-speci fic manner (Mello and Conte, Jr., 2004;Meister and Tuschl, 2004). In brief, the double-stranded RNA is processed by Dicer into small interfering RNA (siRNA) or micr oRNA (miRNA). The 21 nucleotide siRNA and miRNA are then incorporated in the effector complex, RNA-Induced Silencing Complex (RISC), which either cleaves or inhibits translation of the target mRNA In 2005, two key components of RISC, Argonaute2 (Ago2) and siRNA/miRNA, were found to be enriched in GWB (Sen and 35

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Blau, 2005;Pillai et al. 2005;Jakymiw et al. 2005;Liu et al. 2005b;Pauley et al. 2006). MiRNA-targeted mRNA also localizes to GWB in a miRNA-dependent manner (Liu et al. 2005b). These observations provide the first evid ence that RNAi is linked to GWB and opened a new era in our understanding of intracellular RNAi processing. In addition, Ago2 interacts with GW182 in human cells (Jakymiw et al. 2005;Liu et al. 2005a) and this inte raction is conserved in C. elegans and Drosophila (Ding et al. 2005;Behm-Ansmant et al. 2006). Disruption of GWB either by a dominant negative effect or by GW182-knockdown impairs siRNA and miRNA activities (Jakymiw et al. 2005;Liu et al. 2005a), indicating that GW182 and/or GWB are important for RNAi function. Furthermore, miRNA-mediated mRNA degradation requires GWB components such as GW182, the decapping complex Dcp1/Dcp2 and the deadenylase Ccr4:Not (Rehwinkel et al. 2005;Behm-Ansmant et al. 2006), whereas miRNA-mediated translational repression requi res rck/p54 (Chu and Rana, 2006). GWB are highly dynamic structures. First, GW B change in size and number in response to cell proliferation, nutrient conditions and the cell cycle. GW B are larger and more numerous in proliferating cells whereas they are apparently fewer in resting and nutrient starved cells (Yang et al. 2004). During cell cycle, smaller GWB are seen in early S phase and larger GWB are seen in late S and G2 phases. The majority of GWB disassembled prior to mitosis and small GWB reassembled in early G1 (Yang et al., 2004). Second, as sites for the 5 3 mRNA decay, the size and number of GWB are affected by blocking deadenylation, decapping, 5 3 mRNA degradation or tran slation (Sheth and Parker, 2003;Cougot et al. 2004;Andrei et al. 2005;Teixeira et al. 2005). GWB require RNA for assembly and the amount of mRNA or mRNA decay intermediates accumulated in GWB a ffects the size and number of these foci (Sen and Blau, 2005;Brengues et al. 2005;Teixeira et al. 2005). Third, and more interestingly, our 36

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recent studies show that blocking the genesi s of miRNA disassembles GWB and introducing siRNA into these cells re-assembles these foci implicating that either miRNA or the miRNA activities are crucial for the formation of GWB (Pauley et al. 2006). Since siRNA is very similar to miRNA structurally, we were inte rested to determine whether siRNA or siRNAmediated activities also have an effect on the a ssembly of GWB. The answer to this question will help us understand the corr elation between RNAi activity and the formation of GWB. Materials and Methods Antibodies The human prototype anti-GWB (anti-GW182 an d anti-Ago2) sera were obtained from serum banks at the Advanced Diagnostics Laborat ory, University of Calgary. The selection of sera was based on specific reactivity to either GW182 or Ago2 (Jakymiw et al., 2006). Rabbit anti-Ago2 was a gift from Dr. Tom Hobman (U niversity of Alberta, Edmonton, Canada) and rabbit anti-Dcp1a was obtained from Dr. Jens Lykke-Andersen (University of Colorado). Rabbit polyclonal anti-LSm4 was produced as described previously (Eystathioy et al. 2002b). Mouse monoclonal anti-lamin A/C 636, anti-TIAR and an ti-tubulin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), BD Biosci ences (San Jose, CA), and Sigma-Aldrich (St. Louis, MO), respectively. Rabbit polyclonal anti-GFP and anti-rck/p54 were purchased from Invitrogen Corporation (Carlsbad, CA) and MBL International Corporat ion (Boston, MA), respectively. Chicken polyclonal anti-LSm1 wa s purchased from GenWay Biotech Incorporated (San Diego, CA). Small Interfering RNA The siRNAs used in the current study were all purchased from Dharmacon (Lafayette, CO). The siRNAs were dissolved in 1x Univ ersal buffer (provided by Dharmacon) and the resulting 20 stock was stored in aliquots at -20C prior to use. The pre-designed siRNAs 37

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include siCONTROL siRNA for human/mous e/rat lamin A/C (cat# D-001050-01-05), siCONTROL RISC-Free siRNA (cat# D-00122001) and siGENOME SMARTpool siRNA for human RAGE (cat# M-003625-01). The sense and antisense strand of the re st of siRNAs with known sequence are listed below respectively: Individual siGENOME ON-TARGET Human TNRC6 (GW182) siRNA duplex: 5-GAA AU G CUC UGG UCC GCU AUU-3 and 5-P UAG CGG ACC AGA GCA UUU CUU-3 (cat# D-014107-01-0020); hAgo2: 5-GCA CGG AAG UCC AUC UGA A dTdT-3 and 5-UUC AG A UGG ACU UCC GUG C dTdT-3 (Chu and Rana, 2006); hrck/p54: 5-GCA GAA ACC CUA UGA GAU UUU-3 and 5-AAU CUC AUA GGG UUU CUG CUU-3 (Chu a nd Rana, 2006); hLSm1: 5 -GUG ACA UCC UGG CCA CCU CAC UU-3 and 5-GUG AGG UGG CCA GGA UGU CAC UU3 (Chu and Rana, 2006); hLamin A/C: 5P-CUG GAC UUC CAG AA G AAC A dTdT-3 and 5-Cy3-UGU UCU UCU GGA AGU CCA G dTdT-3; Lu ciferase GL2 duplex: 5-C GU ACG CGG AAU ACU UCG A dTdT-3 and 5-U CGA AGU AUU CCG CGU AC G dTdT-3 (cat# D-001100-01-20); EGFP: 5-P GGC UAC GUC CAG GAG CGC ACC-3 and 5-P U GCG CUC CUG GAC GUA GCC UU-3 Construction of Inducible GFP3T3 Fibroblast (TRE-GFP3T3) Cells To establish a reliable 3T3 fibroblast cell line expressing tTA, both constructs (pCAG 20 1 and pUHD10-3 Puro) (Era and Witte, 2000) were transfected into 3T3 cells by Fugene 6 (Roche, Indianapolis, IN) and selected with 1 g/ml puromycin in doxycycline-free medium. Clones, which proliferated in doxycycline-free me dium but died in the presence of doxycycline (1 g/ml) (Sigma, St Louis, MO) and puromycin (1 g/ml) were selected as primary parental doxycycline-regulatory 3T3 cells. The open reading frame of EGFP was amplified by polymerase chain reaction (PCR) using LA-Taq polymerase (TAKARA Bio, Otsu, Japan) from pCX-GFP vector (Ikawa et al. 1995). Primers used were; 5 TGCCGACGCGTGCCACC 38

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ATGGTGAGCAAGG, and 5 -ATAAGAATGCGGCCGCTGAGGAGTGAATTCTTACTT. The PCR fragment was ligated in to the MluI-NotI re striction site of the pTRE2hyg expression vector (Clontech, Palo Alto, CA), which contai ned a tetracycline-responsive element. This vector was introduced into the doxycycline-regulated 3T3 cells by Fugene 6 and selected with hygromycin B (200 g/ml) (Invitrogen, Carlsbad, CA). Doxcycline-dependent expression of EGFP was confirmed by the GFP expression with or without doxycycline (1 g/ml). Cell Culture and Transfection HeLa, HSG, NIH 3T3 and GFP3T3 cells were cultured in DMEM containing 10% fetal bovine serum in a 37C incubator with 5% CO2. SiRNA was transiently transfected into cells grown on glass coverslips in a six-well plate using Oligofectamine (Invitrogen). Briefly, the cultured cells were grown to 30-40% confluency. Then 100 nM or, in the case of co-transfection of two different siRNAs, 100 nM of each siRNA wa s transfected into cells. Usually, cells were fixed 2 days after the transfection. In the 4-day time point experiment, cells were fixed at day 1, day 2, day 3 and day 4 after transfection. In th e sequential transfection experiment, the second siRNA transfection was performed 24 hours after th e initial transfection a nd the cells were fixed at 2 or 3 days after the second transfection. In the plasmid and siRNA co-transfecting experiment, HeLa cells were grown to 50-70%. Then the GFP vector was co-transfected with siRNA either for Ago2, or for rck/p54, or for LSm1 at 1:3 ratio (w/w) using Lipofectamine 2000 (Invitrogen). To test the efficiency of Ago2-knockdown by siRNA, GFP-Ago2 was cotransfected with siRNA for Ago2 at 1:1 ratio (w/w) and then the ce lls were lysed 2 days later. The transfected cells from all the above expe riments were either pr ocessed for indirect immunofluorescence (IIF) or lyse d for western blot analysis. 39

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Western Blot Analysis Cells were harvested in RIPA buffe r (150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl, PH 7.5) containing complete protease cocktail inhibitors (Roche). When whole cells lysate was used to detect the expression level of GWB components upon siRNA transfection, cells were lysed in Laemmli sample buffer directly. Afterwards, equal amounts of protein extract we re separated on 7.5% or 10% polyacrylamide gel and transferred to nitrocellulose. The nitrocellu lose membrane was blocked in 5% non-fat dried milk in PBS-Tween for 1 hour at room temperature, and then probed with primary antibodies to the following proteins for 1 hour: Ago2 (1: 200), Dcp1a (1:1000), rck/p54 (1: 500), LSm1 (1:2000), LSm4 (1:200), Tubulin (1:3000) and GFP (1:200). The membrane was then incubated with horseradish-peroxidase-conjugated goat an tibodies for 1 hour and immunoreactive bands were detected by the Supersignal Chemiluminescent system (Pierce). Fluorescence Microscopy Cells were fixed and permeabilized as previously described (Jakymiw et al. 2005). For colocalization studies cells were incubated at room temper ature with primary antibodies to the following proteins for 1 hour: GW182 (human serum, 1:6000), lamin A/C (1:100), Dcp1a (1:500), rck/p54 (1:500) and TIAR (1:100). Afterwards, cells were incubated with the corresponding secondary fluorochrome-conjugated goat antibodies at room temperature for 1 hour. Alexa Fluor 488 (1:400), Al exa Fluor 568 (1:400), Alexa Fl uor 350 (1:100) (Invitrogen) and Cy5 (1:100) (Jackson ImmunoResearch La boratories, West Grove, PA) were the fluorochromes used. Last, glass coverslips were mounted onto the glass slides using either Vectashield Mounting Medium with or without 4,6-diamidino-2-phenylindole (DAPI, VECTOR Laboratories). Fluorescent images were captured with a Zeiss Axiovert 200M microscope fitted with a Zeiss AxioCam MRm camera using x10 0.75NA, x20 0.75NA, x40 0.75 NA or x63 1.4 40

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NA objectives. All the exposure times and gain settings within one set of experiment are equivalent. Color images were processed us ing Adobe Photoshop (San Jose, CA) version 7. Statistical Analysis GWB/P bodies in each cell were monitored based on light intensity using the Axio Vs40 software (Ver. 4.5.0.0, Zeiss). Images from a comp lete experiment were taken using the same exposure time and about 2 to 3 different areas (100 cells) were rando mly selected for the measurement of the number of GWB using CellP rofiler object counting software program (Carpenter et al. 2006). The threshold was set to a va lue so that the background signal was erased and the quantitated foci were confirmed by being overlaid with the original image. The number of foci in each cell was counted by co rrelating the position of each focus with the area around each nucleus which was defined as the cove rage of a cell. Statistical analysis was performed using Prism 4.0c for Macintosh (Graphpad Software Inc., San Diego, CA). Data between groups were compared using Kruskal-Wallis with Dunns multiple comparison tests or Fishers exact test with Bonfe rroni's correction. For the measurement of RNAi activity, about 70 to110 cells from each data group were randomly selected for the measurement of lamin A/C intensity using the AxioVs40 software. The ar ea from each cell nuclei was selected based on DAPI staining and then switched to lamin A/C stai ning for measurement. The median values of lamin A/C signal in the mock transfected (or lucife rase siRNA-transfected) HeLa cells and in the luciferase siRNA and lamin siRN A sequentially transfected (or lamin siRNA singly transfected) HeLa cells were defined as 0 and 100% si RNA function, respectively. In the sequential transfection experiment, the lamin A/C silencing efficiency of the sample group was calculated based on (median fluorescent intensity in mock group median fluorescent intensity in sample group) / (median fluorescent intens ity in mock group median fluorescent intensity in luciferase siRNA and lamin A/C-siRNA sequentially transf ected group) x 100%. In the co-transfection 41

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experiment the lamin A/C silencing efficiency was calculated based on similar formula: (median fluorescent intensity in lucifera se siRNA group median fluorescen t intensity in sample group) / (median fluorescent intensity in luciferase siR NA group median fluorescent intensity in lamin A/C-siRNA group) x 100%. Results The Size and Number of GWB Increased in Cells Transfected with siRNA Eliciting RNA Silencing of Its Endogenous Target To address the question of how siRNA affects the formation of GWB, we transfected HeLa cells with lamin A/C-siRNA which targets an endogenous mRNA, or luci ferase siRNA, which does not target an endogenous mRNA. Interestingl y, we detected larger and greater numbers of GWB in cells with efficient lamin A/C-knockdown by siRNA than in the mock transfected cells or in the cells transfected with luciferase siRNA (Fig. 3-1AB). The accumulation of rck/p54 in GWB also increased in lamin A/C-siRNA-transfect ed cells (Fig. 3-1A). In comparison, cells transfected with luciferase siRNA had comparable GWB to those in the mock transfected cells (Fig. 3-1A). In addition, another siRNA for a different endogenous target, RAGE ( R eceptor for A dvanced G lycation E nd-product) (Bierhaus et al. 2005), also induced larger and greater numbers of GWB (Fig. 3-2). Notably, the RIS C-free siRNA, a siRNA chemically modified by Dharmacon to lose its silencing ability, is similar to luciferase siRNA in that it did not affect the size or number of GWB either (Fig. 3-1D). Ta ken together, these data suggested that siRNA which elicited RNA silencing of its endogenous ta rget was able to increase the size and number of GWB. Interestingly, the protein expre ssion level of GWB com ponents, including Ago2, Dcp1a, rck/p54 and LSm4, did not increase upon tr ansfection of siRNA fo r lamin A/C (Fig. 31C). This supports a hypothesis that these components were r ecruited to GWB from preexisting or nascent pools of protein upon the tr ansfection of siRNA for lamin A/C. 42

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In addition to HeLa cells, we transfected siRNA for lamin A/C into a different cell line, human salivary gland cell line (HSG ), and a similar increase of GW B was detected (Fig. 3-1E). This demonstrated that the observed effect of si RNA on GWB was not restricted to HeLa cells. As will be shown in subsequent experiments, this siRNA-induced increase of GWB is also observed in mouse cells. To exclude the possibility that the transfected siRNA might act like certain stressor, such as sodium arsenite, which is reported to induce the formation of bot h GWB and SG (Kedersha et al. 2005), we examined the effect of lamin A/C-si RNA on the formation of SG (Fig. 3-3). As a result, only numerous large GWB but no anti-TIAR (a marker pr otein for SG) labeled SG was detected in lamin A/C-siRNA-tr ansfected cells (Fig. 3-3). In comparison, many GWB and SG were observed in the arsenite-tr eated cells, a positive control for the stress response, where GWB and SG are often juxtaposed (Fig. 3-3). This da ta indicated that the siRNA-induced increase of GWB was independent of stress response. Small Interfering RNA Required Endogenous Exp ression of Its Target for Inducing an Increase in Size and Number of GWB To further verify that the siRNA-induced incr ease of GWB is dependent on the presence of the siRNA target, we transfected siRNA for GF P into a mouse fibroblas t cell line (NIH 3T3) engineered to express GFP (GFP3T3) integrally (F ig. 3-4). Mock transfected GFP3T3 cells or NIH 3T3 cells transfected with GFP-siRNA served as controls that either missed the siRNA or the target of siRNA, respectively (Fig. 3-4). Our data showed th at the siRNA for GFP efficiently silenced the expression of its ta rget (Fig. 3-4A, 3-5) and induced a prominent increase of GWB only in the GFP3T3 cells (Fig. 3-4A). By using the CellProfiler object counting software (Carpenter et al. 2006) to quantitate GWB in each cell, we showed that the number of GWB per cell in GFP-siRNA-transfected GFP3T3 cells was significantly higher than that either in the 43

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mock GFP3T3 cells or in the GFP-siRNA-transf ected NIH 3T3 cells (Fig. 3-4B). In addition, the percentage of cells with GWB or with increased GWB in the GFP-siRNA-transfected GFP3T3 cells was also remarkably higher than th ose in the mock GFP3T3 cells and in the GFPsiRNA-transfected NIH 3T3 cells (Fig. 3-4C). In comparison, there was no significant difference between the mock transfected NIH 3T 3 cells and GFP-siRNA-transfected NIH 3T3 in either the number of GWB per cell or the per centage of cells with GWB (or with increased GWB) (Fig. 3-4BC). These data demonstrated that GFP-siRNA i nduced an increase in both the number of GWB and the percentage of cells with GWB only in the 3T3 cells expressing GFP, a finding supporting that the siRNAinduced increase of GWB is target-dependent and may correlate with siRNA-elicited silencing activities. The siRNA-Induced Increase of GWB Started on Day 1 after Transfection and Lasted for at Least 4 Days To determine the temporal increase of GW B induced by siRNA, we performed a 4-day time point experiment and monitored the change s of GWB as well as the accumulation of Dcp1a in GWB at each time point. Interestingly, after transfection of siRNA for lamin A/C, GWB were much larger on day 3 and 4 than on day 1 (Fig. 36A, 3-7A). Quantitative analysis showed that the average number of GWB in lamin A/C-siRNA-transfected cells was higher than that of the mock cells or luciferase siRNA-transfected ce lls through day 1 to 4. The maximal increase of GWB was approximately 5-fold of mock cells a nd occurred on day 3 (Fig. 3-6B, 3-7B). In addition, the percentage of ce lls with GWB in lamin A/C-si RNA-transfected cells (88%%) was higher than that of the other two groups (35%%) through day 2 to 4 (Fig. 3-6C). In comparison, the number of GWB through day 1 to day 4 was similar in luciferase siRNAtransfected cells and the mock tr ansfected cells (Fig. 3-6B). Th e percentage of cells with GWB was also similar between these tw o groups (Fig. 3-6C). Notably, the variation of GWB (in size 44

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and number) during cell cycle was more easily detect ed in these two groups than in the lamin A/C-siRNA-transfected cells (Fig. 3-7 and data not shown). A sim ilar temporal increase in the accumulation of rck/p54 in GWB was observed (Fig. 3-6A and data not shown). Taken together, these data demonstrated that si RNA that elicits mRNA silencing could induce an increase both in the number of GWB and in the percentage of cells with GWB. This increase in number and size of GWB occurred on day 1 after transfection and lasted for at least 4 days. GW182 Was Required for the siRNA-Induced Increase of GWB GW182 is important for both GWB formation (Yang et al. 2004) and miRNA/siRNA activity (Jakymiw et al. 2005;Liu et al. 2005a;Chu and Rana, 2006). We were interested to examine how the knockdown of GW182 affects the siRNA-induced increase of GWB. We used the same GW182-siRNA tested previously to be efficient in silencing the target (Jakymiw et al. 2005). As shown, the transfected GW182-si RNA disassembled GWB and abolished the accumulation of Dcp1a in foci without affecting th e level of lamin A/C expression (Fig. 3-8A). Quantitative analysis confirmed that GW182-siRNAtransfected cells only had 0.22 fold of the GWB in mock cells (Fig 3-8B). More intere stingly, numerous large GWB induced by lamin A/C-siRNA were absent in cel ls where siRNA for GW182 and lamin A/C were co-transfected and where RNA silencing was impaired (Fig. 3-8A). This observation was supported by the quantitative data which indicated the number of siRNA-induced GWB dropped from 3.11 fold to 0.25 fold upon GW182 knockdown (Fig. 3-8B). Luciferase siRNA served as a control siRNA that did not affect the assembly of GWB. In summary, these data indicated that the siRNAinduced increase of GWB required GW182, suggesting that the inte grity of GWB and/or RNAi activity are very important for the siRNA-induced increase of GWB. 45

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The siRNA-Induced Increase of GWB Required Ago2 and Correlated with RNA Silencing Activities To further determine the correlation betw een RNAi activity and the siRNA-induced increase of GWB, we were interested to knockdown Ago2, another GWB component that is a key component of RNA silencing (Liu et al. 2004). The siRNA used in this study was shown to efficiently silence the expre ssion of Ago2 by both another group and us (Fig. 3-9A) (Chu and Rana, 2006). Interestingly, when co-transfecti ng the Ago2 siRNA and the GFP vector at a 3:1 ratio (w/w), we detected discrete foci in the GFP positive cells, which likely contained the siRNA for Ago2 (Fig. 3-9B). Consistently, the number of GW B (labeled by anti-Dcp1a) in Ago2-knockdown cells were only slightly less th an control cells (0.75 fold, Fig. 3-9CE), indicating Ago2 is not essential for the formati on of GWB. To examin e the effect of Ago2knockdown on the siRNA-induced increase of GW B, Ago2 siRNA and la min A/C-siRNA were co-transfected into HeLa cells. As a control, luciferase siRNA was co-transfected with lamin A/C-siRNA. Notably, Ago2-knockd own impaired the silencing function of lamin A/C-siRNA (60% remained, Fig. 3-9D) and abolished the in creased size and number of GWB (labeled by anti-Dcp1a) induced by lamin A/C-siRNA, resulti ng in the number of foci decreasing from 1.77 fold to 0.81 fold (Fig. 3-9CE). Ago2-knockdown also decreased the percentage of cells with foci induced by lamin A/C-siRNA, which dropped to a per centage similar to that of control cells (Fig. 3-9F). In comparison, cells tr ansfected with siRNA for lamin A/C and luciferase had high efficiency of RNA silencing (97.8%) and large nu mbers of prominent foci (Fig. 3-9CD). Taken together, these data indicated that Ago2 was not essential for GWB formation but was required for the siRNA-induced increase of GWB. The observation that knockdo wn of Ago2 inhibited the siRNA-induced increase of GWB strongly s uggested that impairme nt of RNAi function affects GWB dynamics. 46

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Knockdown of LSm1 or Rck/p54 Did not Inhi bit the Assembly of GWB Induced by siRNA To further dissect the correla tion of RNAi activity with the siRNA-induced increase of GWB, we performed knockdown experiments fo r LSm1 and rck/p54, both reported to be important for the formation of GWB but have no effect on siRNA-mediat ed silencing (Chu and Rana, 2006). The siRNA for LSm1 and rck/p54 are shown by us (Fig. 3-10A) and other investigators (Chu and Rana, 2006) to inhibit the expression of its ta rget. To confirm the roles of the LSm1 and rck/p54 in the formation of GWB, we co-transfected a G FP vector with siRNA either for LSm1 or for rck/p54 at a ratio of 1:3 (w/w) into HeLa cells for 2 days (Fig. 3-10B). GFP vector was co-transfected with luciferase siRNA as a control. GWB were barely detected in rck/p54-knockdown cells (Fig. 3-10B). In co mparison, a few small GWB were observed in LSm1-knockdown cells (Fig. 3-10B), implying that LS m1 may be required for the formation of a subset of GWB. In general, both LSm1-knockdown and rck/p54-knockdown prevented the formation of large prominent GWB induced by lamin A/C-siRNA (Fig. 3-10C and data not shown). Nevertheless, sequentially transfecting lamin A/C-siRNA reassembled many small GWB in rck/p54-knockdown cells, resulting in the percentage of cells with GWB increasing drastically from 7% (0.07 GWB/per cell) to 86% (~20 GWB/per cell) (Fig. 3-10C and data not shown). Apparently, the residual rck/p54 was recruited to the newly assembled GWB in rck/p54-knockdown cells, despite the highly efficien t silencing of rck/p54 in these cells (Fig. 310C, 3-11). In LSm1-knockdown cells, sequentially transfecting lamin A/C-siRNA reassembled fewer small GWB (Fig. 3-10C). Furthermore, C y3 labeled lamin A/C-siRNA localized to these reassembled GWB and efficiently silenced the ex pression of its target both in LSm1-knockdown cells and in rck/p54-knockdown cells (Fig. 3-10C). Notably, the localization of Cy3-lamin A/C siRNA to GWB was not only found in Cy3-siRNA strongly transfected ce lls, but also found in Cy3-siRNA weakly transfected cells which was more easily detect ed in rck/p54-knockdown 47

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cells (Fig. 3-10C). Consistent with the above data and data from others (Chu and Rana, 2006), knockdown of LSm1 or rck/p54 did not affect siRNA-mediated silencing (Fig. 3-10D). Most importantly, the assembly of these siRNA-induced GWB correlated with the silencing of lamin A/C both in LSm1-knockdown cells (87%) and in rck/p54-knockdown cells (79%) (Fig. 3-10E). In summary, LSm1 and rck/p54 c ontributed to the formation of GWB to different degrees. However, knockdown of either did not preven t the siRNA-induced assembly of GWB or localization of siRNA to GWB. The siRNA-induced assembly of GWB in LSm1-knockdown cells and in rck/p54-knockdown cells corre lated with RNA silencing activities. Discussion The major and highly reproducible observati on reported in the current study is that siRNA:mRNA induces the appearance of numerous large GWB in the majority of cells where the normal variation of GWB in size and numbe r during the cell cycle was greatly obscured, whereas in untransfected cells large GWB were only detected in a small fraction of cells at late S and G2 stage of the cell cycle. Further study indicated that this siRNA-induced increase in size and number of GWB was regulated by RNAi activity. Our results provide novel insights into the correlations between siRNA function and the as sembly of GWB, suggesting that GWB could serve as markers for siRNA-mediated activity in mammalian cells. Small Interfering RNA:mRNA Initiates Aassemb ly of Microscopic Detectable GWB by Recruiting GWB Components siRNA:mRNA nucleated the assembly of GW B possibly by recruiti ng GWB components. We hypothesize that GWB components are actively exchanged between the cytoplasmic pool and GWB based on the actively ongoing siRNA/ miRNA function and the mediated mRNA decay/translational repression. On e model is that siRNA:mRNA is targeted to or recruits components to pre-existing submicroscopic GWB, which then develop into larger cytoplasmic 48

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structures detectable by conventional microscopy. Alternatively, siRNA:mRNA itself forms de novo GWB by recruiting necessary com ponents/complexes for silencing. The greatest numbers of large GWB were induced on day 3 after siRNA transfection suggesting that the large GWB may be more relate d to siRNA-mediated mRNA decay processes. We postulate that the smaller GWB (or the subm icroscopic GWB), which increased on day 1 or even earlier, may be rela ted to the early stage of RNA sile ncing. The formation of large GWB could be attributed to the si RNA-mediated degradation of la rge amounts of mRNAs, which may have exceeded the maximal capacity of the mRNA decay machinery. As recently proposed, this could then lead to accumulation of these mRNAs or mRNA decay intermediates in GWB (Franks and Lykke-Andersen, 2007). Similarly, increased accumulation of mRNA decay intermediates in GWB due to possible interferen ce by Cy3 dye in the degradation of target mRNA is a reasonable interpre tation for why Cy3-lamin A/C siRNA induces more numerous large GWB than does unlabeled lamin A/C-siRNA with exactly same sequence (data not shown). The Role of GWB Components for the Assembly of GWB Based on the requirement of different GWB components examined in this study for the assembly of GWB, we can deduce some scen arios for GWB assembly. Since GW182 and rck/p54 are important for miRNA-mediated decay/tr anslational repression, their requirement in GWB formation may be attributed, at least in pa rt, to the amount of miRNA-mediated repressed mRNPs maintained in GWB. Notably, GW182-knockdown greatly in hibited the reassembly of GWB induced by siRNA:mRNA resulting in very fe w detectable GWB. This may suggest that GW182 is required at the early stage of GW B assembly. In contrast, rck/p54-knockdown prevented the formation of large GWB but not small GWB induced by siRNA:mRNA suggesting that rck/p54 may function at a late r stage after the initial trigge r of GWB formation. Moreover, rck/p54-knockdown may limit the am ount of mRNPs shuttled to one GWB and the excess RNPs 49

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have to be shuttled to other unsaturated GW B, thereby forming more numerous but smaller detectable GWB. Furthermore, Cy3-lamin A/C siRNA localized to these reassembled small GWB and mediated efficient silencing indicati ng that small GWB are capable of carrying out RNA silencing. It is possible that the RNA decay in small GWB is less efficient than that in large GWB; however, this speculation will need to be addressed in future experiments. Interestingly, even in cells with efficien t rck/p54-knockdown the residua l rck/p54 was detected and concentrated in the reassembled GWB (Fig. 3-11) suggesting that th e recruitment of rck/p54 to GWB is very efficient. This recruitmen t may be via siRNA:mRNA-associated Ago2 since rck/p54 directly interacts with Ago2 (Chu and Rana, 2006). It is possible that rck/p54 contributes to the assembly of these newly formed GWB or it is recruited there for downstream function. Nevertheless, we cannot differentiate whether the asse mbly of these siRNA-induced GWB is required for siRNA-mediated silenci ng or is the consequence of siRNA-mediated silencing. Since almost complete knockdown of rck/p54 barely aff ected RNA silencing efficiency, we postulate that, in general, rck/p54 is not importa nt for siRNA function, unless it efficiently fulfills functions with only residual amounts of the protein. In contrast to GW182 and rck/p54, LSm1 has a less profound effect on the assembly of GWB. The incomplete disassembly of GW B by LSm1-knockdown was previously reported (Andrei et al. 2005) whereas complete disappearance of GWB by LSm1-knockdown was reported in another study (Chu and Rana, 2006). The reasons for this discrepancy can be attributed to different ways of defining foci, different ways of determining cells with knockdown, or different efficiency/sp ecificity of the antibody used to detect foci. Nevertheless, our data are in agreement with the conclusion that LSm1 contributes to th e formation of GWB. The reassembly of GWB induced by siRNA: mRNA in LSm1-knockdown cells implies that 50

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LSm1, like rck/p54, is not required to initiate th e assembly of GWB and is possibly involved in the formation of larger foci. Similar to LSm1, Ago2 has less profound effect on the assembly of GWB compared to GW182 and rck/p54. Knockdown of Ago2 had a mi nor effect on the accumulation of Dcp1a indicating that Ago2 is not required to stab ilize mRNA decay factors in GWB and that the mRNA processing stage could be independent of the siRNA/miRNAmediated silencing stage. Furthermore, the function of A go2 in miRNA-mediated transla tional repression could possibly be compensated by other Argonaute proteins in mammalian cells. This explanation was supported by previous studies where A go2-knockdown did not affect miRNA function profoundly (Chu and Rana, 2006) and where Agos14 had equal capabilit y in binding miRNAs (Meister et al. 2004). Regulation of GWB Assembly An understanding of the regulati on of GWB assembly in mammalian cells has been greatly advanced by the current study. Our data sugge st that, in mammalian cells, the majority of mRNAs degraded via the 5 3 pathway or translationally re pressed in GWB are mediated by siRNA or miRNA. We speculate that under certain circumstan ces GWB may serve as markers for siRNA/miRNA activity and, therefore, the variation in number and size of GWB may correlate with the activities of miRNA during different stages of the cell cycle and proliferation (Yang et al. 2004;He et al. 2005;O'Donnell et al. 2005;Hatfield et al. 2005;Lian et al. 2006). Interestingly, a recent publication reported that Drosophila siRNA:mRNA or miRNA:mRNA also nucleated the formation of GWB (Eulalio et al. 2007b), an observation that strongly supports our finding that siRNA/miRNA-mediated function is a key regulatory mechanism of GWB assembly. Nonetheless, their data also im plicated differences in the regulation of GWB 51

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formation in Drosophila from that in human. For ex ample, Ago2-knockdown disassembled GWB and long dsRNA did not restore GWB in LSm1-knockdown or rck/p54-knockdown cells in Drosophila (Eulalio et al. 2007b). These apparent discrepancies with our data may be attributed to the potential difference in the function of GWB components and in the RNAi pathway between Drosophila and human cells (Okamura et al. 2004;Lee et al. 2004). Depending on the presence of the RNAi mach inery, the regulation of GWB assembly might vary between species. For example, the RNAi machinery as well as related cofactors, such as Argonaute proteins and GW182, are absent in S. cerevisiae. The absence of RNAi in S. cerevisiae may explain the observed differences between yeast P bodies and mammalian P bodies (GWB) in responding to stre sses (Sheth and Parker, 2003;Yang et al. 2004;Brengues et al. 2005;Teixeira et al. 2005). As yeast P bodies are cons idered sites for processing global messages, GWB are more like specific cellu lar structures regulating and organizing siRNA/miRNA-mediated function. This is consistent with the concept that most mRNAs are degraded via the 5 3 pathway in yeast, whereas in mammalian cells only a portion of mRNAs are degraded via the 5 3 pathway while the majority of mRNAs are degraded via the 3 5 by exosome-mediated process (Wilusz et al. 2001;Tourriere et al. 2002;Coller and Parker, 2004;Parker and Song, 2004). The correla tions between GWB and RNAi are proven to be strong. *This work was published in Molecular Bi ology of the Cell, 2007 Sep, Vol 18 (9):3375 87. 52

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Figure 3-1. Transfection of siRNA for lamin A/C increased the size and nu mber of GWB. A) Lamin A/C-siRNA induced an increase in the size and number of GWB (ii compared to i, v compared to iv) in HeLa cells whereas luciferase siRNA did not (iii compared to i, vi compared to iv). HeLa cells were mock transfected or transfected with siRNA for either lamin A/C or luciferase. Cells were fixed 3 days after transfection and stained with index human anti-GWB serum (g reen, i-iii) or rabb it anti-rck/p54 (green, iv-vi) for visualizing GWB and mouse anti -lamin A/C to monitor the knockdown of lamin A/C (blue, i-vi). Scale bar, 10 m. B) Western blot anal ysis demonstrated that siRNA for lamin A/C achieved efficient gene silencing. The level of tubulin 53

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reactivity served as a loadi ng control. C) Expression of components of GWB was not apparently affected upon transf ection of siRNA either for lamin A/C or luciferase. HeLa cells were transfected with 100 nM siRNA either for lamin A/C or luciferase and lysed 3 days later. The whole cell lysa tes were analyzed by western blot for the expression of GWB components includi ng Ago2, Dcp1a, rck/p54, and LSm4. The level of tubulin reactivity se rved as a loading control. D) Transfection of RISC-free siRNA had no detectable effect on GWB (green, iv compared to i) as luciferase siRNA (green, iii compared to i). HeLa cells were mock transfected (i, v) or transfected with 100 nM lamin A/C-siRNA (ii, vi), or luciferase siRNA (iii, vii), or RISC-free siRNA (iv, viii). Cells were fixed on day 3 after transfection and stained with human anti-GWB serum for GWB (gr een), mouse anti-lamin A/C for detecting the knockdown of lamin A/C (magenta) and DAPI for nuclei (blue). The average number of GWB per cell is shown with the to tal number of cells counted indicated in parentheses. Scale bar, 10 m. E) Transfection of lamin A/C-siRNA induced more GWB (iii-iv compared to i-ii, ix-x compared to vii-viii) in human salivary gland (HSG) cell line on both day 3 and 4 after transfection whereas luciferase siRNA did not (v-vi compared to i-ii, xi-xii compared to vii-viii). The HSG cells were mock transfected or transfected w ith 100 nM siRNA either for lamin A/C or luciferase. The transfected cells were fixed 3 and 4 days after transfection and then stained with human anti-GWB serum (magenta) and rabbi t anti-Dcp1a (green) for GWB, mouse anti-lamin A/C (blue) for detecting th e knockdown of lamin A/C. The average number of GWB per cell is shown with the to tal number of cells counted indicated in parentheses. Scale bar, 10 m. 54

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Figure 3-2. Transfection of siRNA for RAGE activated assembly of GWB. RAGE-siRNA was transfected into HeLa cells to examin e its effects on GWB formation. Mock transfected cells or cells transfected with siRNA for luciferase served as controls. Cells were fixed on day 3 after transfecti on and stained with human anti-GWB serum (green) and counterstained with DAPI (b lue). The number and size of GWB increased only in cells transfected with siRNA for RAGE (ii). Scale bar, 10 m. 55

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Figure 3-3. Lamin A/C-siRNA di d not induce stress granules. Lamin A/C-siRNA induced numerous large GWB (viii compared to ii) but did not induce SG (vii compared to i and iv). In contrast, sodium arsenite induced SG (iv compared to i) and an increase of GWB (v compared to ii). An enlarged cell section is shown in the bottom right corner for arsentie-treated cells (iv-vi), il lustrating that SG were often adjacent to GWB (inset of vi). The cells were count erstained with human anti-GWB (magenta), mouse anti-TIAR for SG (green) and DAPI fo r nuclei (blue). Merged image of each row are shown in the right column. Scale bar, 10 m. 56

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Figure 3-4. The siRNA-induced in crease of GWB is target-depe ndent. A) GFP-siRNA induced an increase in the size and number of GWB in GFP3T3 cells (iv compared to i) but did not in NIH 3T3 cells (vii compared to i). Expression of GFP is efficiently inhibited in GFP-siRNA-tran sfected GFP3T3 cells (gree n, v compared to ii). The cells were fixed on day 3 after mock transf ection or transfection of GFP-siRNA, and were co-stained with human anti-GWB serum (magenta, i, iv, vii) for GWB and DAPI for nuclei (blue). Merged image of each row is shown in the right column. Scale bar, 10 m. B-C) Quantitative analyses indi cated that GFP-siRNA induced an increase both in the number of GWB per cell and in the percentage of cells with GWB in GFP3T3 cells. The number of GWB per cell was counted in 100 to 200 cells for each group. Each dot represented a single cell and was plotted on a graph with the number of GWB per cell as Y-axis (B). Th e median with the interquartile range is indicated for each data group. *, signifi cant difference between groups indicated by bracket (Dunns multiple comparison test, P<0.001); ns, no significant difference between groups indicated by bracket (Dunns multiple comparison test, P>0.05). 57

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Based on the data from (B), the percentage of cells with GWB (open bars) or increased GWB (filled bars) was calculated and shown for each individual group(C). Seven foci per cell, the median value of GFP-siRNA-transfected GFP3T3 group, are set as the standard value to define cells with increased GW B. The bars indicated by are significantly higher than the others (Fishers exact test, P<0.001). 58

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Figure 3-5. Green fluorescence protein (GFP) was efficien tly knocked down by siRNA. GFP3T3 cells were either mock transfected (iii, iv) or transfected with 100 nM siRNA for GFP (i, ii), and then fixed 3 days later. Nuclei were counterstained with DAPI (blue). Scale bar, 10 m. 59

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Figure 3-6. The increases of GWB started on da y 1 and were most prominent on day 3 after transfection of siRNA for lamin A/C. A) HeLa cells were mock transfected, or transfected with 100 nM siRNA for lamin A/C or for luciferase, and then fixed on day 1 (i-vi), day 2 (vii-xii), day 3 (xiii-xviii) and day 4 (xix-xxi v) after transfection. Cells were counterstained with human anti-GW B serum (magenta) and rabbit anti-Dcp1a (green) to monitor the cha nges of GWB. The level of lamin A/C was evaluated by using mouse anti-lamin A/C (blue). Scale bar, 10 m. B) Quantitative analysis indicated that cells transfected with la min A/C-siRNA had a significant increase in the average number of GWB per cell thr ough day 1 to day 4. The number of GWB per cell was quantitated as described in Fig. 3-4 and Me thods. The groups indicated by are significantly higher than the other groups on the same day (Dunns multiple comparison test, P<0.001). C) The percentage of cells with GWB in lamin A/CsiRNA-transfected cells (filled square) is significantly higher than mock cells (filled circle) and luciferase siRNA-transfected cells (filled triangle) through day 2 to 4 60

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(Fisher exact test, P<0.001). In comparis on, the other two groups were very similar to each other (Fisher exact test, no significant difference from day 1 to 3, P>0.05). 61

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Figure 3-7. The increase of GWB induced by la min A/C-siRNA was prominent on day 3. HeLa cells were either mock transfected (i) or transfected with 100 nM siRNA for lamin A/C (ii) or luciferase (iii), and then fi xed on day 3 after transfection. Cells were counterstained with index human anti-GWB serum (green) to monitor GWB and DAPI for nuclei (blue).A) A majority of th e cells transfected with siRNA for lamin A/C had larger and greater numbers of foci. Scale bar, 10 m. B) Quantitative analysis of the number of foci per cell show ed that both the percentage of cells with foci and the average number of foci per ce ll increased in cell treated with lamin A/CsiRNA. The number of foci per cells was quantitated as described in Fig. 3-4B and methods. The median with the interquartile range is indicated for each data group. The lamin A/C-siRNA group, as indicated by bracket, was significantly higher than the other two groups (Dunns multiple comparison test, P<0.001); ns, no significance (Dunns multiple comparison test, P>0.05). 62

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Figure 3-8. GW182 was required for the siRNA-induced increase of GWB. A) GW182knockdown inhibited both the increase of GWB (x-xi compared to xiii-xiv) and the lamin A/C-knockdown (xii, compared to xv) induced by lamin A/C-siRNA. HeLa cells were transfected with 100 nM si RNA for GW182 (iv-vi), 100 nM siRNA for lamin A/C (vii-ix), or both (x-xii) for 3 days SiRNA for luciferase in both the single siRNA transfection (i-iii) and co-transfection (xiii-xv) served as c ontrols. Cells were stained with human anti-GWB serum (magen ta) and rabbit anti-Dcp1a (green) for GWB, mouse anti-lamin A/C for monitoring lamin A/C-knockdown (b lue). Scale bar, 10m. B) Quantitative analysis showed that knockdown of GW182 abolished the siRNA-induced increase of GWB. The numbe r of GWB in ~300 cells collected from 3 randomly selected fields was counted fo r each group. The average number of GWB per cell in each group was divided by that of mock cells to calculate the fold difference. The bars indicated by are si gnificantly higher th an others (Fishers exact test, P<0.001). 63

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Figure 3-9. The siRNA-induced increase of GWB required Ago2 and correlated with RNA silencing activities. A) West ern blot analysis showed th at siRNA for Ago2 efficiently knocked down Ago2. GFP-Ago2 was co-transf ected with siRNA either for Ago2 or for lamin A/C at 1:1 ratio (w/w) for 2 days Tubulin reactivity served as a loading control. B) Ago2-knockdown ba rely affected the formation of GWB. SiRNA for Ago2 (i-ii) or luciferase (iii -iv) was co-transfected with GFP vector (green) at 3:1 ratio (w/w) into HeLa cells for 2 days. Th e cells were counterst ained with rabbit antiDcp1a (red). In cells transfected with A go2 siRNA, discrete GWB were detected in GFP-positive cells (i-ii, arrows), which was comparable to the GWB in GFP negative cells in the same panel (i-ii, arrowhea d) or to the GWB in GFP-positive cells transfected with luciferase siRNA (iii-iv, arrows). Scale bar, 10 m. C-F) SiRNAinduced increase of GWB was abolishe d when Ago2 was knocked down and RNA silencing efficiency was impaired. HeLa cells were transfected with 100 nM siRNA for Ago2 (i, ii), 100 nM siRNA for lamin A/C, or both (v, vi) for 2 days. SiRNA for luciferase in both the single siRNA transfec tion and co-transfection (iii, iv) served as controls. Cells were stained with hu man anti-GWB serum (red) and rabbit anti64

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Dcp1a (green) for GWB, DAPI for nucl ei (blue), mouse anti-lamin A/C for monitoring lamin A/C-knockdown (lamin A/C staining is not shown). C) Ago2 siRNA diminished the increase of GWB induced by lamin A/C-siRNA. Fewer GWB were observed in cells co-transfected w ith siRNA for Ago2 and lamin A/C (v, vi) than cells with siRNA for both luciferase and lamin A/C (iii, iv). Scale bar, 10 m. D) Quantitative analysis of lamin A/C silencing efficiency indicated that Ago2knockdown impaired RNAi activity. AxioV40 software was used to measure the fluorescent intensity of nuclear lamin A/C staining in each cell for each group (70 cells per group). The median value of la min A/C fluorescent intensity in each group was used to calculate its corresponding RNA silencing efficiency based on the formula described in Methods. Y axis lamin A/C silencing efficiency. E) Quantitative analysis showed that Ago2-knockdown significantly decreased the average number of GWB induced by siRNA. The number of GWB in each cell was counted as described in Fig. 3-4B and Met hods. Then the resulte d average number of GWB per cell in each group was divided by that of control cells to calculate the fold difference. The bars indicated by are si gnificantly higher th an others (Fishers exact test, P<0.001). F) Ago2-knockdown decrea sed the percentage of cells with GWB induced by siRNA. Based on the data fr om (E), the percentage of cells with GWB was calculated and shown for each individual group. The bars indicated by are significantly higher than othe r groups (Fishers exact test, P<0.001). 65

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Figure 3-10. Knockdown of LSm1 or rck/p54 disassembled GWB but did not inhibit the assembly of GWB induced by siRNA. A) Knockdown of LSm1 or rck/p54 in HeLa cells by siRNA. HeLa cells were tran sfected with siRNA for LSm1 or rck/p54, harvested 2 days later and analyzed by western blot with antibodies to LSm1 or rck/p54. Tubulin reactivity served as a load ing control. B) Rck/p54 is required for the assembly of majority of GWB whereas LSm1 is important only for a portion of GWB. GFP vector were co-transfected with siR NA for LSm1, rck/p54, or luciferase at 1:3 ratio (w/w) into HeLa cells for 2 days. Th e transfected cells were counterstained with 66

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human anti-GWB serum (magenta) and rabbit anti-rck/p54 (green). Merge images of anti-GWB and anti-rck/p54 are shown in the right column. The average number of GWB per cell and the percentage of cells with GWB are shown with the total number of cells counted indicated in parentheses. Scale bar, 10 m. C) Lamin A/C-siRNA induced the assembly of GWB and locali zed to these GWB in spite of LSm1knockdown or rck/p54-knockdown. 100 nM si RNA either for LSm1 (iv-vi) or rck/p54 (vii-xii) were transfected into HeLa cells and 24 hours later 100 nM Cy3 labeled lamin A/C-siRNA (red) were sequentia lly transfected. The transfected cells were fixed 3 days after the 2nd transfection and stained with human anti-GWB serum (green), mouse anti-lamin A/C (magenta) and DAPI (blue). Merged images of Cy3siRNA and anti-GWB are shown in the ri ght column. The percentages of cells exhibiting a similar or iden tical GWB staining to the ce lls presented are shown in panel iv and x with the total number of cells counted indicated in parentheses. Panels iv-ix show representative localization of siRNA to GWB in Cy3-siRNA strongly transfected cells whereas panels x-xii show the localization in weakly transfected cells. Insets are enlarged by 1.5 to 2 folds and the Cy3 signal is enhanced to show the localization of siRNA to GWB. Arrows indicate the co -location for the weak Cy3 signals in enlarged insets. Arrowheads indicate the co-l ocalization for the strong Cy3 signals in GWB. Scale bar, 10 m. D) Knockdown of LSm1 or rck/p54 did not affect RNA silencing activities. AxioV40 softwa re was used to measure the fluorescent intensity of nuclear lamin A/C staining in each cell for each group (~100 cells per group). The median value of lamin A/C fluorescent intensity in each group was used to calculate its corresponding RNA sile ncing efficiency based on the formula described in Methods. Y axis, lamin A/ C silencing efficiency. E) The siRNAinduced assembly of GWB correlated with RNA silencing activities. One hundred to 150 cells from each group are randomly selected and subjected to quantitation according to the assembly of GWB and th e knockdown of lamin A/C. GWB +, cells exhibiting similar or identical GWB staining to the cells presented in panel C, iv for LSm1-knockdown or panel C, x for rck/p54-knockdown; GWB -, cells without microscopic detectable GWB; Lamin KD +, the fluorescent intensity of nuclear lamin A/C staining is lower than 50% of th e median value of lamin A/C fluorescent intensity of the mock cells; Lamin KD -, the lamin A/C intensity is higher than 50% of the median value of lamin A/C intensity of mock cells. 67

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68 Figure 3-11. Lamin A/C-siRNA induced assembly of GWB and recruited residual rck/p54 to the reassembled GWB in rck/p54-knockdown cells HeLa cells were either mock transfected (i-iii) or transf ected with 100 nM siRNA for rck/p54 (iv-vi) for 3 days. In the sequential transfection, 100 nM lamin A/ C-siRNA were transfected 24 hours after the initial transfection of si RNA for rck/p54 and the cells were fixed 2 days after that 2nd transfection (vii-ix). The transfected cells were stained with human anti-GWB serum (green), rabbit antirck/p54 (red), mouse anti-lamin A/C (magenta) and DAPI (blue). Merge image of anti-rck/p54 and an ti-GWB are shown in the right column. Insets are enlarged about 1.5-fold and the rck/p54 signal in rck/p54-knockdown cells (vii, ix) is enhanced to show the localization of rc k/p54 to GWB. Arrowheads indicate co-localizat ion. Scale bar, 10 m.

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CHAPTER 4 THE C-TERMINAL HALF OF AGO2 BINDS TO MULTIPLE GW-RICH REGIONS OF GW182 AND REQUIRES GW182 TO MEDIATE SILENCING Introduction MicroRNA (miRNA)-mediated gene silencing is an important post-transcriptional regulation which controls in part the half lives of mRNA targets. In this regulation, miRNA binds to the 3-UTR of mRNA leading to translational inhibition, mRNA degradation, and mRNA sequestration (Nilsen, 2007). MiRNAs are evolutionarily c onserved in most of species and are estimated to regulate ~30% of protein-encoding genes in human (Lewis et al. 2005;Filipowicz et al. 2008). There are four human Ago proteins that include Ago1 to Ago4, which are the core components of silencing effecter complexes and are known to bind single-stranded miRNA. These Ago proteins share greater than 80% iden tity and are primarily characterized by PAZ and PIWI domains. The PAZ domain contains a bi nding pocket for the 3 overhanging nucleotides of miRNA. Interestingly, despite highly c onserved sequences, only the PIWI domain of Ago2 harbors RNase H-type activity and, therefore, Ago2 also functions in siRNA-mediated slicing of mRNA targets (Liu et al. 2004;Yuan et al. 2005). Tethering Ago prot eins to the 3-UTR of mRNA mimicked miRNA function and effected translational repression (Pillai et al. 2004). GW182 is important for miRNA-mediated transl ational silencing and interacts with Ago2. GW182 contains several glycine/tr yptophan-rich (GW-rich) regions a glutamine/asparagine-rich (Q/N-rich) domain, and a C-terminal R NA recognition motif (RRM) (Eystathioy et al. 2002a;Decker et al. 2007). GW182 localized to and was essential for the formation of GW bodies (GWB, also known as mammalian P bodies) (Yang et al. 2004;Schneider et al. 2006), cytoplasmic structures closel y linked to mRNA decay (Sheth and Parker, 2003;Eystathioy et al. 2003b) and the miRNA/siRNA pathway (Jakymiw et al. 2005;Pauley et al. 2006;Lian et al. 69

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2006;Lian et al. 2007). Knockdown of GW182 greatly impaired miRNA-mediated gene silencing and subsequent mRNA degradation (Rehwinkel et al. 2005;Liu et al. 2005a). Interestingly, Ago proteins, miRNAs, and mRNAs targeted by miRNAs all colocalized with GW182 in GWB (Sen and Blau, 2005;Pillai et al. 2005;Jakymiw et al. 2005;Liu et al. 2005b). Furthermore, GW182 interacted with Ago2 and this interaction was conserved from plants to human (Ding et al. 2005;Jakymiw et al. 2005;Liu et al. 2005a;Behm-Ansmant et al. 2006;ElShami et al. 2007;Till et al. 2007). However, the role of GW182 and the importance of GW182-Ago2 interaction in translational repression remain unclear. In the current study, we mapped the GW182-Ago2 interaction and investigated the possible role of this interaction in miRNA-mediated silencing in human. Materials and Methods Construction of Deletion Co nstructs of GW182 and Ago2 The details of constructing GW1 1 (aa254, formerly known as GW182 1), Ago2 (aa1 860) and PIWI (aa478 860) were described previously (Jakymiw et al. 2005). To construct pENTR-TNR (aa1 204), PCR amplification was c onducted on the human testis cDNA (BioChain) using primer 5TTT GGA AGA TCT ATG AGA GAA TTG GAA GCT AAA GCT-3 and primer 5-AAG GGA AGT GCC ATT CAT ACC-3, which is downstream of an internal Kpn I site (nt1252). The 1.5kb PCR product was digested with Bgl II and Kpn I to generate a 1.2kb fragment that wa s used to replace the 5 500bp BamHI-Kpn I fragment of pENTR-GW182. Afterwards, the pENTR-GW182 with the extra N-terminal ~750nt was digested with BamHI (nt610) and Not I (3 end linker) to releas e a 6.5kb fragment comprising a majority of GW182. The vector fragment contai ning the N-terminal region was ligated at room temperature (RT) for 1 hour to ge nerate pENTR-TNR (aa1). GW1 1a (aa254 503) was 70

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constructed by PCR using primer 5 -GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CAA CGC CAT GGA TGC TGA TTC T-3, and primer 5 -GGG GAC CAC TTT GTA CAA GAA AGC TGG GTG GGA AGT GCC ATT CAT ACC TG-3. Annealing temperature was 55.7oC for first 2 cycles and then 62.5oC for additional 30 cycles. GW1 1b (aa502 751) was constructed by PCR using primer 5 -GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CAC TTC CCT TTC TCA CCT TAG CA-3, a nd primer 5-GGG GA C CAC TTT GTA CAA GAA AGC TGG GTG GCC TCT GTC CCA TTG TCA GT-3. Annealing temperature was 55.2oC for the first 2 cycles and then 62.4oC for additional 30 cycles. GW1 7 (aa10341962) was constructed by PCR using primer 5 -GGG GAC AAG TTT GT A CAA AAA AGC AGG CTT CAA AGA CCA GCA AGC ACA GGT ACA3, and primer 5 -GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT AGG CAA CAT CAA GGC AT A G-3. Annealing temperature was 55.7oC for the first 2 cycles and 63.3oC for additional 30 cycles. The human Ago3 mutant (Ago3m) in pCMV-SPORT6 vector wa s obtained from Invitrogen (Clone number: CS0DB008YP10). Ago3m sequence was amplified by PCR using primer 5-GGG GAC AAG TTT GTA CAA AAA AGC AGG C TT CAT GGA AAT CGG CTC CGC AGG ACC C-3, and primer 5GGG GAC CAC TTT GTA CAA GAA AGC TGG GTA TCA GAC CTT GGC CCC CAC A-3. Sequences, GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CA and GGG GAC CAC TTT GTA CAA G AA AGC TGG GT, common in the above forward and reverse primers were designed to incorporate the recombin ation sites. The products from the above PCR reactions were then cloned into pDONR207 (Inv itrogen) using the Gateway BP recombination reaction as per the manufacturers instructi ons (Invitrogen). To construct pENTR-GW1 5 (aa16701962), pENTR-GW182 was digested with Sal I (5 end linker) and Spe I (nt5008) to release a 4.2Kb vector fragment containing the C-terminus of GW182. The overhangs of this 71

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fragment were filled in and then li gated. To construct pENTR-MGW (aa566 1343), phrGFPKIAA1460 was digested with XhoI (5 end linker) and Sma I to release a 2.3Kb fragment, which was then subcloned into the Sal I and EcoR V sits of pENTR2B (Invi trogen). To construct pENTR-PAZ (aa1480), pENTR-Ago2 was digested with XhoI (nt1467) and XhoI (3 end linker) to generate the vector fragment containing N-terminal half of Ago2, which was purified and then ligated. To construct pENTR-Ago1, ES T clone pBluescript hAgo1 was first digested with BamHI (3 end linker) and the overhang was filled in to generate a blunt 3 end. Then the digested product was cut by Kpn I (5 end linker) to generate a 4.0 kb fragment which was subcloned into the Kpn I and EcoR V sites of pENTR1A (Invitroge n). To construct pENTRAgo4, EST clone pBluescript hAgo4 was digested with Sma I (5 end linker) and ScaI (3 end linker) to generate a 3.5 Kb fragment which was then subcloned into the Dra I and EcoR V sites of pENTR1A (Invitrogen). All of the variants used in curr ent study were subcloned into Gateway compatible GST, GFP or 3xFlag vect ors by using Gateway LR recombination reaction (Invitrogen). pIreSneo-Flag/HA Ago3 was obt ained from Thomas Tuschl (Meister et al. 2004) through Addgene. The tethering assay plasmi ds including pClneo-NHA vector, NHA-Ago2, Renilla luciferase RL-5BoxB and FL were gifts from Dr. Witold Filipowicz, Friedrich Miescher Institute for Biomedical Research, Switzerland (Pillai et al. 2004). To generate NHA-PIWI (aa478) and NHA-PAZ (aa1), pCIneo-NHA vector was converted to gateway destination vector using the Gateway Vector Conversion Syst em (Invitrogen). Then PIWI (aa478) and PAZ (aa1) were moved fr om corresponding pENTR vectors to the pCIneo-NHA gateway vector respec tively by recombination. All DNA constructs used in this study were confirmed by direct DNA sequencing. 72

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Antibodies Rabbit anti-Ago2 and rabbit anti-GST were gifts from Dr. Tom Hobman (University of Alberta, Edmonton, Canada) and Dr. Peter Sayeski (University of Florida, Gainesville, USA), respectively. Mouse monoclonal anti-HA was purchased from Covance (Emeryville, CA). Mouse monoclonal anti-Flag M2 and anti-tubulin were purchased from Sigma-Aldrich (St. Louis, MO). Rabbit polyclonal anti-GFP was purchased from Invitrogen Corporation (Carlsbad, CA). Plasmid Transfection, GST Pull-do wn, and Western Blot Analysis HeLa cells were cultured in DMEM c ontaining 10% fetal bovine serum in a 37oC incubator with 5% CO2. HeLa cells were grown to 90-100% in 6-well plat e at the day of transfection. GST-tagged constr uct was singly transfected or co -transfected with other tagged constructs into HeLa cells us ing Lipofectamine 2000 (Invitrogen ) as per the manufacturers instructions for 24 h. For detection of expressi on of GST-tagged proteins in whole cell lysate, the cells were lysed in Laemmli sample buffer di rectly. For GST pull-dow n assay, the cells were lysed with NET/NP40 buffer (150mM NaCl, 5m M EDTA, 50mM Tris, pH 7.4, 0.3% NP40) with Complete Protease Cocktail Inhibitor (Roche Di agnostics) and then sonicated at 20% amplitude for 10 sec for 3 times on ice. Afterwards, the lysates were centrifuge d at 13,200 rpm for 5 min. The pellets (insoluble fractions) were lysed in Laemmli sample buffer directly. The soluble fractions were incubated with Glutathione SepharoseTM 4B (GE Healthcare) and mixed at 4oC for 2 h for GST pull-down. After the incubati on, the beads were washed with NET/0.3% NP40 buffer for four times and the samples eluted in Laemmli sample buffer. The soluble fraction of cell lysates (input), GST pull-down samples, whol e cell lysates, and inso luble fractions were separated on 10% polyacrylamide gel and transfer red to nitrocellulose. Western blotting was performed as described previously (Lian et al. 2007). The dilutions of primary antibodies were: 73

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1:1000 for anti-GST, 1:400 for anti-Flag, 1:1000 for anti-GFP, and 1:500 for anti-Ago2, 1:1000 for anti-HA. Indirect Immunofluorescence Cells were fixed and permeabilized as described previously (Jakymiw et al. 2005;Lian et al. 2007). The dilution of anti-Flag antibody was 1:1000. Tethering Assay Using a Du al Luciferase System HeLa cells were grown to about 90~100% c onfluence in 24-well plate at the day of transfection. To determine the effect of tethering PIWI (aa478 860) and PAZ (aa1 480), the cells were transfected with 0.1ng of constructs expressing reporter Re nilla luciferase (RL5BoxB), 100ng of control firefly luciferase (FL) plus 700ng of NHA tag, NHA-Ago2, NHAPIWI, or NHA-PAZ using Lipofectamine 2000 (Invitrogen) for 48 h as per the manufacturers instructions. Cells were harves ted 48 hour after tran sfection and the FL and RL activities were measured using Dual-Luciferase Reporter Assay System (Promega, Madison, WI) following the manufacturers protocol. Relative luciferase ac tivities (ratio of targeted luciferase activities over control luciferase activities) were calculated as described previously (Pillai et al. 2004). Briefly, FL/RL activity in NHA vector transf ected (control) group was regarded as 0% translational repression. The repr ession levels of other experime ntal groups were calculated by the percentage reduction of relative luciferase activities compared to that in NHA control group. The assay was performed in triplicates and repeated for 2 to 3 times. To detect the expressions of tethered NHA-tagged proteins, the above cell lysates were mi xed at 1:1 ratio with Laemmli sample buffer and western blot was performed as described in Western Blot Analysis of Materials and Methods. 74

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RNA Interference and Quantitative Real Time PCR The sequence of siRNA for GW182 or for GFP was described previously (Lian et al. 2007). To determine how GW182-knockdown affect s the Ago2or PIWI-mediated suppression, cells were grown to 30-50% confluence and we re transfected with 100 nM of siRNA for GW182, or siRNA for GFP as a negative control using Lipofectamine 2000 (Invitrogen). Thirty hours later, these cells were tr ansfected again with constructs expressing reporter RL-5BoxB, control FL and NHA tagged constructs as described above. Fortyeight hours after the second transfection, total RNA was extracted from HeLa cells using RNeasy Mini Kit (Qiagen, Valencia, CA). RNase-Free DNase Set (Qiagen) was applied to eliminate potential DNA contamination. The relative mRNA level of GW182 was measured in duplicate using Ct method (Livak and Schmittgen, 2001) and TaqMan Fast Universal Master Mix (Applied Biosystems) with the corresponding TaqM an Gene Expression Assay (TNRC6A, Hs00379422_m1, Applied Biosystems). The level of 18S rRNA was measured as internal control (18S rRNA, 4310893E, Applied Biosystems ). The melting curve in each individual measurement was monitored to guard against non-specific amplification. Results C-terminal Half of Human Ago2 Containing the PIWI Domain Was Responsible for the Interaction with GW182 The interaction between human Ago2 and GW182 was first reported in 2005 (Jakymiw et al. 2005;Liu et al. 2005a). To further characterize th is interaction, a se ries of deletion constructs covering different domains of GW18 2 and Ago2 were generated (Fig. 4-1A). The human GW182 gene identified as TNRC6A in the GenBank database is currently predicted to have 2 isoforms: GW182 as prev iously described (Eystathioy et al. 2002a) and a longer alternative-spliced product (NM _014494.2) that contains an extr a N-terminal 253aa-polypeptide 75

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with a glutamine-repeat (Q-repeat) region enco ded by CAG trinucleotide repeats (TNR). The deletion construct TNR (aa1 204) included the Q-repeat regi on and did not overlap with GW182 (Fig. 4-1A). TNR (aa1) and ot her two GW182 deletion constructs GW1 1 (aa254 751) and MGW (aa5661343), respectively representing appr oximately the N-terminal 1/3 and middle 1/3 of GW182, were initially used to analyze the binding to Ago2 using the GST pull-down assay. GST-GW1 1 and GST-MGW were demonstrated to interact with endogenous Ago2 whereas GST-TNR did not (Fig. 4-1B). To map the region of Ago2 responsible for this interaction, two deletion constructs corresponding to the Nterminal (PAZ, aa1 480) and Cterminal (PIWI, aa478 860) halves were constructed. By co-expression of GSTand Flagtagged constructs and the GST pull-down assa y, Ago2 consistently interacted with GW1 1 and MGW but not with TNR (Fig. 4-1C). Important ly, PIWI but not PAZ was responsible for the association of Ago2 with GW1 1 and MGW (Fig. 4-1C). Nota bly, there was less of GST-MGW in the soluble input when it was co-transfected with Flag-Ago2 or Flag-PIWI than when GSTMGW was co-transfected with Flag-PAZ (Fig. 4-1C). To investigate the reason for these observed differences, the expres sion of GST-MGW was examined in both total cell lysates and the insoluble fractions. The expression of GST-MGW in total cell lysates was relatively uniform no matter which Flag-tagged construc ts it co-transfected with (F ig. 4-2). Interestingly, higher levels of GST-MGW was observed in the insolu ble fraction when it was co-transfected with Flag-Ago2 and even higher when co-transfected with Flag-PIWI, whereas GST-MGW was barely detectable when co-transfected with Fl ag-PAZ. This data suggested that MGW formed insoluble complexes with Ago2 or PIWI, which might explain why there was a lower level of MGW in the soluble input for the GST pulldown assays. In summary, GW182 fragments 76

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GW1 1 and MGW both bound to Ago2 and this binding wa s mediated by the C-terminal half of Ago2. GW182-Ago2 Interaction Was Important for the Localization of Ago2 in Cytoplasmic Foci Previous studies have shown that Ago2 colo calized with GW182 in cytoplasmic GWB and GW182 was essential for the formation of these foci (Yang et al. 2004;Jakymiw et al. 2005). However, the driving force for the localizat ion of Ago2 to GWB remains unknown. Based on the above data that the GW182 fragment MGW formed insoluble complexes with Ago2 and PIWI, but not with PAZ, we hypothesized that the GW182-Ago2 interaction is crucial for Ago2 to localize to GWB. To examine this hypothesis, Flag-Ago2, -PIWI or -PAZ was co-expressed with GFP-MGW in HeLa cells and Flag-PIWI or -PAZ were expressed alone as controls (Fig. 43). Flag-Ago2 was shown to colocalize with GFP-MGW in cytoplasmic foci whereas singly expressed Flag-PIWI or -PAZ were diffusely distributed in the cy toplasm (Fig. 4-3). Interestingly, co-expression of GFP-MGW with Fl ag-PIWI dramatically changed the distribution of Flag-PIWI, which was recru ited to cytoplasmic foci and co localized with GFP-MGW (Fig. 43). In contrast, co-expressing GFP-MGW with Flag -PAZ did not recruit th e diffusely distributed Flag-PAZ to cytoplasmic GFP-MGW-positive foci. This data supported th at the contention that interaction of GW182 with the C-terminal half of Ago2 mediated the lo calization of Ago2 in GWB. Ago2 Bound to Multiple Non-overla pping GW-rich Regions of GW182 Since the GW182 fragments, GW1 1 and MGW, were both shown to bind Ago2 and these two fragments have overlapping 186aa, it is possible that the overlapping region of GW182 (aa566) is the primary site for the GW182-Ago2 interaction. To examine this possibility, deletion constructs GW1 1a (aa254 503) and GW1 1b (aa502751) were generated with the 77

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latter covering the overlapping region of GW1 1 and MGW (Fig. 4-1A). In addition, other deletion constructs GW1 7 (aa1034 1962) and GW1 5 (aa16701962) were used to investigate whether regions of GW182 other than GW1 1 and MGW bound Ago2. GFPGW1 1a, -GW1 1b, -GW1 7, or -GW1 5 was co-expressed with GS T-tagged Ago2 fragment PIWI in HeLa cells and a GST pull-down assay wa s performed to examine the interaction. As a negative control, GFP-GW1 1 was co-expressed with GST-tagge d fragment N1, the N-terminal aa51 of a completely unrelat ed protein hZW10 (Famulski et al. 2008). Unexpectedly, GFP-GW1 1a, -GW1 1b, -GW1 7, and -GW1 5 all co-precipitated with GST-PIWI (Fig. 4-4). Interestingly, GW1 1a, GW1 1b, and GW1 5 are non-overlapping fragment s and thus this data showed that at least 3 separate regions of GW182 could bind Ago2. Moreover, the Ago2binding deletion constructs all co ntain GW-rich region whereas TN R, the only deletion construct that did not bind Ago2 lacked a GW-rich region. In summary, the GW182 deletion constructs containing GW-rich regions all bound to the C-terminal half of Ago2 indicating that multiple regions of GW182 mediated the in teraction of GW182 with Ago2 and that GW repeat might be an key element for Ago2-binding. The Interaction of Ago2 with GW182 Was Conserved in Other Human Ago Proteins There are four Ago proteins in human that sh are a high degree of se quence similarity. To examine whether Ago1, Ago3, and Ago4 interact with GW182, GFP-Ago1, -Ago3, -Ago3m, or Ago4 was co-expressed with GS T-tagged GW182 fragments GW1 1 or MGW in HeLa cells and a GST pull-down assay was performed. Ago3m is a splicing variant of Ago3 and is missing aa757 823, the C-terminal 66aa of PIWI domain. Interestingly, human Ago1, Ago3, and Ago4 bound GW1 1 and MGW (Fig. 4-5). Ago3m did not bind GW1 1 or MGW indicating that the C-terminus of PIWI domain was required for the binding to GW182 (Fig 4-5). Notably, both 78

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GFP-Ago1 and Flag-Ago1 bound GST-GW1 1 demonstrating that different N-terminal fusion tags did not affect the bindi ng of Ago1 with GW182. In summary, the interaction of human GW182 with Ago2 was observed with other human Ago proteins and the C-terminal region of PIWI domain was critical for th e interaction of GW182 with Ago3. Tethering C-terminal Half of Ago2 to the 3-UTR of mRNA Recapitulated Ago2-mediated Silencing Which Required GW182 It was reported that tetheri ng Ago2 to the 3-UTR of mRNA causes repression of protein synthesis (Pillai et al. 2004). Since GW182 and Ago2 are stably associated with each other, the GW182-Ago2 interaction might help Ago2 mediate silencing through interaction with 3-UTR of mRNA. Because the C-terminal half of Ago2 was shown to bind GW182 whereas the Nterminal half of Ago2 was not, we examined whet her the C-terminal half of Ago2 was able to mediate silencing when tethered to the 3-UTR of mRNA. The dual lu ciferase and tethering assay was used as descri bed previously (Pillai et al. 2004). In this assay, the reporter Renilla luciferase (RL) contains five 19-nt BoxB hairpin structures in the 3-UTR of its mRNA (RL5BoxB). The N peptide, which is derived from phage and binds to BoxB structures with high affinity (Legault et al. 1998), was fused to the N-terminus of Ago2, PIWI, and PAZ. In this way, Ago2, PIWI, or PAZ was brought directly to the 3-UTR of mRNA bypassing the requirement for miRNA. Interestingly, teth ered PIWI was attended by almost as much repression as tethered full-length Ago2 was (Fig. 4-6A). In cont rast, tethered PAZ was totally devoid of the repression function of Ago2 (Fig. 4-6A). This data indicated that the functional domain mediating silencing lie w ithin the C-terminal half of Ago2. To examine whether GW182 is required for Ago2or PIWI-mediated re pression, siRNA was used to knockdown GW182 before Ago2, PIWI or PAZ was tethered to the reporter mRNA. Very interestingly, both Ago2and PIWI-mediated repression was significantly reduced upon GW182-knockdown (Fig. 4-6B). 79

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The GW182-knockdown was confirmed by quantitative real time PCR (Fig. 4-6C). In summary, tethering the C-terminal half of Ago2 to th e 3-UTR of mRNA recap itulated the repression function of Ago2 and this repression required GW182. Discussion Formation of GW182 and Ago Protein Complexes Two studies have identified th at one GW-rich region capable of binding Ago2 is conserved in the plant and yeast orthologs of GW182, and that the GW repeat within th is region is critical for GW182-Ago2 interaction (El-Shami et al. 2007;Till et al. 2007). Consistent with these studies, our data showed that human GW-rich regions were important for Ago2-binding (Fig. 47). GW182 fragments MGW (aa566) and GW1 7 (aa1034) containing the ortholog-conserved GW-rich region (aa1074) (Till et al. 2007) were shown to bind Ago2 (Fig. 4-1A). In addition, our data showed that at least three non-overl apping regions of GW182 could independently bind Ago2 and, interestingl y, these Ago2-binding fragments are outside of the ortholog-conserved GW-rich re gion (Fig. 4-7). Sequence ali gnment analysis showed that 27aa and 23aa residues of the ortholog-cons erved GW-rich region shared 40.7% and 34.8% identity with the GW1 5 and GW1 1b, respectively (Fig. 4-7). However, significant sequence identity was not identified between the ortholog-conserved region and GW1 a. The precise amino acid requirement for Ago2-binding remains unclear and requires fu rther investigation. Nevertheless, our data lead to the speculati on that one GW182 protein can bind multiple Ago proteins and this may contribute to the formation of functional tran slational silencing complexes. Since GW182 interacted with all four Ago protei ns, it is possible that different Ago proteins incorporate into the same complex. The func tion of the silencing complex might depend on which Ago proteins it contains. It was reported more closely-sp aced miRNA binding sites in the 80

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3-UTR of target mRNA lead to more effi cient miRNA-mediated translational repression (Grimson et al., 2007). This supports our speculation th at GW182 helps to stabilize the binding of multiple Ago-miRNA complexes to the 3-U TR of target mRNA for more efficient translational repression. It is also possible that GW182 simu ltaneously binds to Ago-miRNA complexes on several different mRNAs and this GW182-Ago interaction may be the driving force for the assembly of submicroscopic and mi croscopic GWB. This hypothesis is supported by current observations that the GW1 1 GW182 fragment or the PIWI Ago2 fragment could mediate GW182-Ago2 interac tion and by our previous data that overexpression of either of these two constructs disassembled GWB, possibly due to disruption of GW 182-Ago2 interaction by a dominant-negative effect (Jakymiw et al., 2005). C-terminal Half of Ago2 Preserved the Silencing Function of Ago2 Probably Because It Maintained the Interaction with GW182 Our GW182-Ago2 interaction mapping showed that the C-terminal half of Ago2 (aa478) was sufficient for the binding with GW182 whereas N-terminal half (aa1) was not required. Interestingly, only the C-terminal half of A go2 preserved the silencing function of Ago2 when directly brought to th e 3-UTR of target mRNA. The s ilencing function mediated by Ago2 or the C-terminal half of Ago2 was abol ished upon GW182-knockdown. Our data strongly suggested that interactio n of Ago2 with GW182 is critical fo r the silencing process mediated by Ago2 at the 3-UTR of target mRNA. This hypot hesis is also supported by two recent studies where overexpressing the Ago-bi nding fragment of yeast or Drosophila ortholog of GW182 greatly disrupted GW182-Ago inte raction and significantly impai red miRNA-mediated silencing in vitro and in vivo (Till et al. 2007;Eulalio et al. 2008). Our data also suggested that Ago2 is not the f inal repressor because its silencing function relied greatly on GW182. GW182 probably functions downstream of Ago proteins and mediates 81

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translational repression one step further. Interestingly, the PIWI domain of Ago2 was reported to be responsible for the interaction with Dicer (Tahbaz et al. 2004). It is intriguing to postulate that Dicer and GW182 may compete for the bi nding with Ago2 through the PIWI domain. Notably, tethering the N-terminal half of Ago2 to the 3-UTR of mRNA seemed to upregulate protein levels. A previous study showed that te thering the PAZ domain alone or the smaller Nterminal fragment of Ago2 to the 3-UTR of mRNA did not upregulat e translation (Pillai et al. 2004). This discrepancy may be explained by the difference in th e deletion constructs used in these studies. Our data lead to the speculation that there may be an activation domain in the N-terminal half of Ago2 that could upregul ate protein synthesis and may explain how Ago2miRNA complex can activate translation unde r certain circumstances (Bhattacharyya et al. 2006;Vasudevan et al. 2007;Buchan and Parker, 2007). Ho wever, defining this activation domain and how it activates transla tion needs further investigation. Based on the data from current study, we propose a model for miRNA-mediated gene silencing in which, after miRNA guides Ago-miR NA complex to the 3-UTR of target mRNA, Ago protein recruits GW182 to stabilize Ago-miRNA-mRNA bindi ng and represses translation. In addition, GW182-Ago2 interaction recruits Ago2 to GWB, which accumulate many Ago2miRNA-mRNA complexes and become cente rs for miRNA-mediated silencing. *This work was submitted to Journal RNA for publication and is in revision now. 82

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Figure 4-1. Identifying the interac tion of C-terminal half of Ago2 with GW182 fragments using GST pull-down assays. A)Schematic of human GW182 and Ago2 deletion constructs. All amino acid residues are re ferenced to the longer isoform of GW182 (GenBank Accession NM_014494.2). Q-repeat, glutamine repeat (box in white); Q/N-rich, glutamine/asparagine-ric h region (box in magenta); RRM, RNA recognition motif (box in green); GW-rich, glycine/tryptophan-rich region (boxes in yellow); N-GW, N-terminal GW-rich re gion; M-GW, middle GW-rich region; CGW, C-terminal GW-rich region. Human Ago2 is mainly comprised of two domains: PAZ domain (box in blue) and PIWI domai n (box in red). B) Endogenous Ago2 coprecipitated with GW182 fragments GW1 1 (aa254) and MGW (aa566) but not with TNR (aa1 204). GST-tagged TNR (lanes 1,4), GW1 1 (lanes 2,5), or MGW (lanes 3,6) was transfected into HeLa cells. Endogenous Ago2 was detected by using rabbit anti-Ago2. In a longer expos ure (lanes 7), Ago2 was detected more clearly being co-precipitated with GST-MG W (lane 9) but still absent in GST-TNR precipitates (lane 7). C) GW182 fragments GW1 1 and MGW co-precipitated with Ago2 fragment PIWI (aa478) but not with PAZ (aa1). GST-MGW (lanes 1) or GST-GW1 1 (lanes 7) was co-transfected with Flag-tagged Ago2, PIWI, or PAZ. Full-length Ago2 (lanes 4,9) a nd PIWI (lanes 5,10), but not PAZ (lane 6), 83

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co-precipitated with GST-MGW or GST-GW1 1. D) GW182 frag ment TNR did not pull-down Ago2. Flag-Ago2 was co-transfected with GST-tagged TNR (aa1 204, lanes 1, 3) or with GW1 1 (lanes 2, 4) as a positive control. 84

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Figure 4-2. The GW182 fragment MGW formed insoluble complexe s with Ago2 and C-terminal half of Ago2. GST-MGW was co-transfected with Flag-Ago2, -PIWI, or -PAZ in HeLa cells. The expression of GST-MGW in total cell lysate was relatively uniform (lanes 1). Higher level of GST-MGW was detected in the insoluble fraction when co-transfected with Flag-A go2 (lane 4) or Flag-PIWI (lane 5) whereas GST-MGW was barely detectable when co-transfected with Flag-PAZ (lane 6). The level of tubulin served as a loading control. 85

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Figure 4-3. GW182 fragment MGW r ecruited Ago2 to cytoplasmic fo ci by interacting with the C-terminal half of Ago2. GFP-MGW (green, a-c) was co-transfected with Flag-Ago2 (d), PIWI (aa478860, f) or PAZ (aa1 480, h) into HeLa cells. As controls, FlagPIWI (e) or Flag-PAZ (g) was singly transf ected. The cells were stained with antiFlag antibody (red, d-h). Panels in the bo ttom row are the merged images (i-m). Nuclei were counterstained with DAPI (blue). Scale bar, 10 M. 86

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Figure 4-4. The C-terminal half of Ago2 bound to multiple non-overlapping GW-rich regions of GW182. GST-PIWI (aa478) was co -transfected with GFP-GW1 1 (lane 1), GW1 1a (lane 2), -GW1 1b (lane 3), -GW1 7 (lane 4), -TNR (lane 11), or -GW1 5 (lane 12) into HeLa cells. Similar to positive control GW1 1 (lane 6), GW1 1a (lane 7), GW1 1b (lane 8), GW1 7 (lane 9), GFP-GW1 5 (lane 14), but no GFP-TNR (lane 13), were detected in GST-PIWI precipitates. GST-tagged N1, N-terminal fragment from an unrelat ed protein hZW10, was co-t ransfected with GFP-GW1 1 (lane 5) as a negative control and no interaction was detected (lane 10). 87

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Figure 4-5. Both GW182 fragments GW1 1 and MGW co-precipitated with other human Ago proteins. A) Ago 1 and Ago4, but not Ago3 mutant, co-precipitated with GW182 fragments GW1 1 and MGW. GFP-Ago1, -Ago3m (Ago3 mutant), or -Ago4 was co-transfected with GST-MGW (lanes 1) or GST-GW1 1 (lanes 7). Ago3m is missing aa757823, the C-terminal 66aa of the PIWI domain. Both Ago1 (lanes 4,10) and Ago4 (lanes 6,12) were pulled down by GST-MGW or GST-GW1 1. In comparison, Ago3m was absent from eith er pull-down (lanes 5,11). B) Ago3 coprecipitated with GW182 fragment GW1 1. Flag-Ago3 (lanes 1,3) or -Ago1 (lanes 2,4) was co-transfected with GST-GW1 1 (lanes 1). GFP-Ago3m (lanes 5 6) was co-transfected with GST-GW1 1 as a negative control. 88

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Figure 4-6. Translational repre ssion mediated by tethered C-terminal half of Ago2 required GW182. A) Tethered PIWI (aa478) downregulated protein synthesis. HeLa cells were transfected with constructs e xpressing the RL-5BoxB reporter, control FL reporter, and indicated NHA-tagged proteins Bar graphs represent normalized mean values of RL/FL activities with standard errors. The RL/FL values in cells with tethered NHA were normalized as 1. Th e expression of fusion proteins was determined by Western Blot using anti-HA mAb and are indicated below the bar graphs. The assay was performed in triplicates and was repeated for at least 3 times. *significant difference (unpaired t te st, p<0.01); ns, no significant difference (unpaired t test, p>0.05). B) Translational repression me diated by tethered Ago2 or PIWI (aa478) was greatly impaired upon GW182-knockdown. HeLa cells were transfected with siRNA for either GW182 (s iGW182) or GFP (siGFP). Thirty hours later, cells were transfected again with constructs expressing reporter RL-5BoxB, control FL reporter, and the same NHAtagged proteins as indicated in panel A Bar graphs represent the reduction of RL/FL in cells with tethered NHA-Ago2 or NHAPIWI compared to those in cells with tether ed NHA. The reduced values of RL/FL in cells transfected with GFP-siRNA were set as 1. Error bars indicate standard errors. The assay was performed in triplicates and was repeated for 2 times. *significant difference (unpaired t test, p<0.01) C) GW182-knockdown by siRNA was confirmed by quantitative real time PCR. The bar graphs represent normalized mRNA level of GW182 with standard errors The mRNA level of GW182 in cells transfected with GFP-siRNA was set as 1. The experiment was performed in triplicates. 89

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90 Figure 4-7. At least three non-overlapping GW-rich regions that are different from the orthologconserved GW-rich region can independen tly bind Ago2. The dot graph on top indicates the distribution of tryptophan in GW182. W, every tryptophan (magenta diamond); WG/GW, a glycine right adjacent to tryptophan (red triangle); AW/WA, an alanine right adjacent to tryptophan (blue diamond); W only, no glycine or alanine right adjacent to tryptophan (green diamond). The majority of the tryptophans are adjacent to either a glycine or alanine. The schematic of GW182 is indicated below the dot graph. GW-rich region, box in ye llow; ortholog-conserved GW-rich region (aa1074, (Till et al. 2007)) box in red. GW1 1a, GW1 1b and GW1 5 are the three non-overlapping re gions identified in the curr ent study that binds Ago2. The amino acid sequence alignment between GW1 5/ GW1 1b and orthologconserved GW-rich region was performed us ing ExPASy website tool. indicating identical conservation.

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CHAPTER 5 DISCUSSION AND CONCLUSIONS GW/P Body Is a Processing Center for Me ssenger RNAs Targeted by RNAi Pathway with Its Component GW182 Playing a Critical Role in the Silencing Process Two interesting studies (Chu and Rana, 2006;Eulalio et al. 2007b) have been published after our report showing that th e integrity of microscopic-dete ctable GWB is dispensable for RNAi (Jakymiw et al. 2005). In these two studies, disruption of GW B by knocking down decapping factor LSm proteins, which are importa nt for GWB formation, does not affect siRNAor miRNA-mediated silencing. Nevertheless, one of the studies showed that blockage at any step of siRNA or miRNA pathway leads to disappear ance of GWB and that transfecting siRNA into Drosha-knockdown cell refo rms the foci (Eulalio et al. 2007b). These data are consistent with another study from our laboratory (Pauley et al. 2006) and support that formation of GWB is the consequence of siRNAand miRNA-mediated sile ncing. Furthermore, the current studies show that the number and size of GW B increase when siRNA silencing is activated. Knockdown of Ago2 impairs siRNA silencing and prevents th e increase of GWB. Interestingly, although knockdown of rck/p54 or LSm1 disassembles GWB, they do not prevent the reformation of GWB since the siRNA silencing activity is intact (Lian et al. 2007). Altogether, these recent studies indicate that, although the microscopic-detectable GW B are not required for the initiation of siRNA/miRNA silencing, siR NA:mRNA or miRNA:mRNA duplex activates the formation of GWB as long as the RNAi pathway is intact, su pporting that GWB are st ructures for storing and/or processing the sequestered mRNAs targeted by siRNA or miRNA. This is consistent with the observed high enrichment of siRNAs, miRNAs, and target mRNAs in GWB. It is unclear whether the siRNA:mRNA and miRNA:mRNA duplexes require submicroscopic GWB for docking, or they could recruit decay factors to form de novo GWB. Why GWB form and whether the formation of GWB ma kes the subsequent mRNA degrada tion step more efficient are 91

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unknown. To address these possibili ties and questions needs further investigations. It is noted that a recent study report an individual siRNA for CD81, a transmembrane protein apparently unimportant for GWB formation, surprising ly causes disassembly of GWB (Serman et al. 2007). However, other two different siRNAs for the sa me protein do not. This suggests that this particular siRNA may have off-ta rget effects and may have targ eted important genes in RNAi pathway. Since the importance of GWB for RNAi is controversial, we reexamined how disruption of GWB impairs RNAi function in our initial study (Jakymiw et al. 2005) based on the data from the current studies. The three methods we us e to disrupt GWB, in cluding expression of dominant negative constructs GW1 1 and Ago2 PIWI (aa478), and knockdown of GW182, all apparently interfere with the GW182-Ago2 interac tion that has been proven important for miRNA function in human by the current studies and in Drosoph ila by two other studies (Till et al. 2007;Eulalio et al. 2008). The interaction of GW182 w ith Ago2 possibly recruits GW182 to target mRNA to critically repress translat ion. In addition, the GW 182-Ago2 interaction may recruit RISC/miRNP-bound target mRNAs to GWB for processing or cause them to aggregate into GWB. The disassembly of GWB is pr obably the consequence of disruption in GW182Ago2 interaction and impairment in miRNA function. The implication from our initial study is that, potentially, the GW182-Ago2 interaction is required for siRNA-mediated silencing. However, the exact function of this interaction in siRNA function needs to be further confirmed and investigated. Working Model and Conclusions We propose a working model that illustrates the important advance in understanding how GW182 and GW/P body involved in RNAi attribut ed by our current work (Fig. 5-1). Our 92

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findings define for the first time GW182 as a pot ential translation repressor working downstream of Ago proteins. The GW182-Ago interaction may contribute to the aggreg ation of translation repressed mRNAs and the formation of GWB. Although the formation of GWB may not be required for initial RNAi activity, active RNA s ilencing initiates the assembly of GWB which therefore become processing centers for mRNA ta rgets and biomarkers for RNAi activity. The work presented here advance the molecular a nd cell biology of RNAi, a nd may provide insight into the future applicatio n and monitoring of RNAi. GW/P Body May Regulate Multiple Cellular Processes Factors involved in several different cellular pathways have been id entified as components of GWB (Tab. 5-1). In addition to mRNA d ecay/storage/transportation and RNAi, recently GWB have been suggested to associate with viral life cycle and inna te anti-viral defense (Beckham and Parker, 2008). The initial evidence from the analys is of yeast retrotransposons Ty1 and Ty3 shows that yeast P body component LSm1-7p complex and Dhh1p (ortholog of rck/p54) are required for effi cient retrotransposition for both Ty1 and Ty3 (Griffith et al. 2003). In addition, deletion in the deadenylase complex Ccr4/Pop2 results in enhanced retrotransposition (Irwin et al. 2005). Since in yeast LSm1-7p and Dhh1p promote the formation of P body, and Ccr4p/Pop2p limits P body formation (Coller and Parker, 2005;Teixeira and Parker, 2007), these observations s uggest that targeting of Ty transcripts to P bodies might be important for retrotranspositi on. Another evidence is that the human RNA helicase DDX3 is required for the export and tr anslation of unspliced HIV-1 RNA from the nucleus (Yedavalli et al. 2004). Interestingly, the yeast or tholog of DDX3, Ded1p, is shown to accumulate in P bodies and is important for their formation (Beckham et al. 2008). This suggests that in human cells DDX3 may recruit the unspliced HIV-1 RNA, which serves as genomic RNA of HIV-1, to GW/P body for subsequent steps in viral function. Furthermore, 93

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GWB are also linked to anti-v iral defense by the evidence th at the anti-viral proteins APOBEC3G and APOBEC3F are concentrated in GWB, and that APOBEC3G interacts with Ago1 and Ago2 (Wichroski et al. 2006;Gallois-Montbrun et al. 2007). Members of APOBEC (apolipoprotein B RNA-editing enzyme cataly tic polypeptide 1-like) family of cytidine deaminases are thought to play an anti-viral ro le by preventing host cell genome from invasion by retroviruses or retrotransposons (Wedekind et al. 2003). Interestingly, the Vif protein of HIV-1, which binds to APOBEC3G and triggers its degradation, is found to localize to GWB in an APOBEC3G-dependent manner (Wichroski et al. 2006). These observations suggest that APOBEC3G and APOBEC3F might function in the microenvironment of GWB to restrict HIV-1 replication. As important components of GWB, miRNAs has b een shown to play a critical role in cell cycle, cell proliferation and tumor genesis (Lian et al. 2006;Kent and Mendell, 2006;Carleton et al. 2007). More than 10 miRNAs have been identifie d to target factors, such as Bcl-2, Ras, and E2F1, which are critical for cell cycle progr ession and cellular pr oliferation (O'Donnell et al. 2005;Kent and Mendell, 2006). Mo reover, proto-oncogene c-Myc or tumor suppressor p53 is shown to regulate the leve l of specific miRNAs (He et al. 2005;O'Donnell et al. 2005;He et al. 2007a;He et al. 2007b). Alteration of miRNA levels can contribute to pathological conditions, including tumorigenesis, which are associated with loss of cell cycle control (Kent and Mendell, 2006). Interestingly, GWB have been shown to vary in number and size at different stage of cell cycle and during cell proliferation (Yang et al. 2004). All these eviden ce support that GWB are potentially important sites for miRNA-mediat ed regulation of these cellular mechanisms. Two recent studies from our laboratory sugge st that GWB may be associated with autoimmune disease. The first study reports th at a set of autoantibod ies from patients with 94

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rheumatic diseases and in a mouse model of autoimmunity recognize key components of RNAi including Ago1-4 and Dicer (Jakymiw et al. 2006). It implicates th e potential involvement of RNAi pathway in the pathogenesis of autoimmune diseases. This is al so supported by another recent study which shows that misregulation of a single miRNA leads to systemic lupus (Yu et al. 2007). The second study from our laboratory shows that miRNA regulats human monocyte functions such as cytokine and chemokine produ ction via the formation of GW/P body (Pauley et al. 2008). The overproduction of inflammatory cy tokines and chemokines have been shown to closely related with autoimmune diseases such as systemic lupus erythematosus, Sjgrens syndrome and rheumatoid arthritis. The latest advance of RNAi pathway is the discovery of endogenous siRNAs (esiRNAs) in Drosophila and mouse. Investigators used to th ink that esiRNAs only existed in organisms that possess RNA-dependent RNA polymerases (RDRPs), such as plants, C. elegans, and yeast. However, seven studies published recently totally change this concept (Czech et al. 2008;Kawamura et al. 2008;Tam et al. 2008;Watanabe et al. 2008;Ghildiyal et al. 2008;Okamura et al. 2008a;Okamura et al. 2008b). These studies uncover that esiRNAs surprisingly exist in both Drosophila and mouse oo cytes, and that the esiRNAs play a role in suppressing the expression of retrotransposons and in regulating spec ific protein-encoding transcripts complementary to them (Tam et al. 2008;Watanabe et al. 2008). Most of the identified esiRNAs are derived from a variety of sources: long hairpin structures, convergent transcription, bidirectional tran scription, and transposable elements (Nilsen, 2008). Interestingly, some of the esiRNAs in mouse oocytes are pr ocessed from overlapping regions of encoding genes and cognate pseudogenes (Tam et al. 2008;Watanabe et al. 2008). These findings suggest that pseudogenes may actually regulate the expression of their founder gene. Although 95

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the extent and biological relevance of esiRNAs await further invest igation, discovery of esiRNAs as a new class of small RNAs broadens the scope of regulatory networks mediated by small RNAs. Since both Drosophila and mouse th at do not have RDRPs can generate esiRNAs, it is not surprising that esiRNAs will be identified in human cells. It would be interesting to further investigate the possible existence of a nd the potential functions of esiRNAs in human cells. Since esiRNAs are structur ally the same as exogenous siRNA, we speculate that esiRNAs may be also enriched in GWB as siRNA, and that their functions closely associate with GWB as well. Taken together, GWB are directly or indire ctly involved in multip le cellular pathways (Fig. 5-2). It is intriguing to postulate that the driving force for these pathways correlating with GWB is that certain steps of them may be regulated by siRNA or miRNA. The formation of GWB physically sequesters the regulated mRNAs from polysomes which may help to quickly and efficiently stop translation. The translation repressed mRNAs are stored and degraded in the GWB, and may go back to transl ation under certain circ umstance. However, the exact function of GWB in these pathways needs further investigations. 96

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Table 5-1. Protein co mponents of GW/P body Pathway Factors Cell function Reference 5-3 mRNA decay pathway Dcp1:Dcp2 LSm1-7 Xrn1 Ccr4 Ge-1/Hedles Rck/p54 Edc3 Decapping enzyme Stimulate decapping 5-3 exonuclease Deadenylation Enhance decapping Decapping; translation control and repression Decapping activator (van Dijk et al. 2002) (Ingelfinger et al. 2002) (Bashkirov et al. 1997) (Cougot et al. 2004) (Yu et al. 2005) (Chu and Rana, 2006) (Fenger-Gron et al. 2005) ARE-mediated decay pathway TTP, BRF-1 Shuttle ARE-mRNAs to GWB for silencing (Franks and LykkeAndersen, 2007) Nonsensemediated decay pathway Upf1,3 Smg5,7 Shuttle and degrade aberrant mRNA in GWB Dephosphorylate Upf1 (Durand et al. 2007) (Durand et al. 2007) Translation eIF4E eIF4E-T RAP55 Ded1p (yeast) Translation control; regulate cell cycle and proliferation Translation repression; increase mRNA instability Translation control Translation initiation (Andrei et al. 2005) (Culjkovic et al. 2006) (Ferraiuolo et al., 2005) (Yang et al. 2006) (Beckham et al. 2008) siRNA/miRNA pathway GW182 TNRC6B Ago2 Ago1,3,4 Translation repression Translation repression Translation repression; cleave mRNA targets Translation repression (Jakymiw et al. 2005) (Meister et al. 2005) (Jakymiw et al. 2005) (Pillai et al. 2005) Transport and storage of mRNA Nuclear RNA export factor 7 Staufen FMRP Sort, transport, and store mRNA RNA-binding protein; traffic mRNA Translation control; traffic mRNA (Katahira et al. 2008) (Moser et al. 2007) (Moser et al. 2007) Innate antiviral defense APOBEC3G,3F Vif (HIV-1) Inhibit replication of retrovirus APOBEC3 inhibitor (Wichroski et al. 2006) (Gallois-Montbrun et al. 2007) 97

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Figure 5-1. A proposed model of the function of GW182 and GW/P body in RNAi. In siRNA pathway (upper left), siRNA bound to Ago2 fo rming active RISC which then binds to and cleaves target mRNA with complete complementary sequence. The interaction of GW182 with Ago2 recruits the cleaved mRNA to GW/P body for degradation. In miRNA pathway (upper right), miRNA binds Ago protein and forms miRNP, which subsequently binds to the 3-UTR of mRNA with incomplete complementary sequence (boxes in orange on mRNA). However, the binding of miRNP to mRNA is not able to stop translation until GW182 is recruited. GW182 possibly help to stabilize the binding of miRNPs to the 3-UTR and is the repressor for translation. The translation repressed mRNA is recr uited to GW/P body for storage and/or degradation. Under certain circumstance, the stored repressed mRNA can go back to translation. The GW182-Ago protein inter action helps the cleaved and translation repressed mRNAs aggregate into GW/P body where mRNA decay factors (Pacman) are recruited to degrade the messages. U nder the circumstance that siRNAand/or miRNA-mediated silencing are activated, mo re target mRNAs are recruited to GWB for processing leading to an increase in the number and the size of GWB. 98

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99 Figure 5-2. Multiple cellular pathways associated with GWB. The image in the middle is an immunogold electron microscope image of GW body. Arrows point to the cellular pathways that are considered to be associat ed with GWB. Dotted arrows point to the two cellular processes that have been shown regulated by miRNA silencing. The key references proving the links between th ese pathways and GWB are listed as following: miRNA silencing (Liu et al. 2005b), siRNA silencing (Jakymiw et al. 2005), mRNA decay (Sheth and Parker, 2003), mRNA storage (Brengues et al. 2005), viral life cycle (Beckham and Parker, 2008), anti-viral defense (Wichroski et al. 2006;Gallois-Montbrun et al. 2007), autoimmune disease (Jakymiw et al. 2006), cell proliferation (Yang et al. 2004), cell cycle (Lian et al. 2006;Kent and Mendell, 2006), and innate immune signaling (Pauley et al. 2008).

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LIST OF REFERENCES Anderson,P. and Kedersha,N. (2006). RNA granules. J. Cell Biol. 172, 803-808. Andrei,M.A., Ingelfinger,D., Heintzmann,R., Achsel,T., Rivera-Pomar,R., and Luhrmann,R. (2005). A role for eIF4E and eIF4E-transporte r in targeting mRNPs to mammalian processing bodies. RNA. 11, 717-727. Assaad,F.F., Tucker,K.L., and Signer,E.R. (1993) Epigenetic repeat-induced gene silencing (RIGS) in Arabidopsis. Plant Mol. Biol. 22, 1067-1085. Bashkirov,V.I., Scherthan,H., Solinger,J.A., Buerstedde,J.M., and Heyer,W.D. (1997). A mouse cytoplasmic exoribonuclease (mXRN1p) with prefer ence for G4 tetraplex substrates. J. Cell Biol. 136, 761-773. Beckham,C., Hilliker,A., Cziko,A.M., Noueiry,A ., Ramaswami,M., and Parker,R. (2008). The DEAD-Box RNA Helicase Ded1p Af fects and Accumulates in Saccharomyces cerevisiae PBodies. Mol. Biol. Cell 19, 984-993. Beckham,C.J. and Parker,R. (2008) P bodies, stress granules, and vi ral life cycles. Cell Host. Microbe 3, 206-212. Behm-Ansmant,I., Rehwinkel,J., Doerks,T., Stark, A., Bork,P., and Izau rralde,E. (2006). mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes Dev. 20, 1885-1898. Bhanji,R.A., Eystathioy,T., Chan,E.K., Bloch,D.B ., and Fritzler,M.J. (2 007). Clinical and serological features of patients with au toantibodies to GW/P bodies. Clin. Immunol. 125, 247256. Bhattacharyya,S.N., Habermacher,R., Martine,U ., Closs,E.I., and Filipowicz,W. (2006). Relief of microRNA-mediated translational repressi on in human cells subjected to stress. Cell 125, 1111-1124. Bierhaus,A., Humpert,P.M., Morcos,M., We ndt,T., Chavakis,T., Arnold,B., Stern,D.M., and Nawroth,P.P. (2005). Understanding RAGE, the recep tor for advanced glycation end products. J. Mol. Med. 83 876-886. Boerjan,W., Bauw,G., Van,M.M., and Inze,D. (1994). Distinct phenotypes generated by overexpression and suppression of S-adenosyl-L-m ethionine synthetase reveal developmental patterns of gene silencing in tobacco. Plant Cell 6, 1401-1414. Brengues,M., Teixeira,D., and Parker,R. (2005) Movement of eukaryotic mRNAs between polysomes and cytoplasmic processing bodies. Science 310, 486-489. 100

PAGE 101

Buchan,J.R. and Parker,R. (2007). Molecular biology. The two faces of miRNA. Science 318, 1877-1878. Carleton,M., Cleary,M.A., and Linsley,P.S. (2007) MicroRNAs and cell cy cle regulation. Cell Cycle 6, 2127-2132. Carpenter,A.E. et al. (2006). CellProfiler: image anal ysis software for identifying and quantifying cell phenotypes. Genome Biol. 7, R100. Chu,C.Y. and Rana,T.M. (2006). Translation Re pression in Human Cells by MicroRNA-Induced Gene Silencing Require s RCK/p54. PLoS. Biol. 4, e210. Coller,J. and Parker,R. (2004). Eukaryotic mRNA decapping. Annu. Rev. Biochem. 73 861-890. Coller,J. and Parker,R. (2005). General transl ational repression by activators of mRNA decapping. Cell 122, 875-886. Conti,E. and Izaurralde,E. (2005). Nonsense-mediated mRNA decay: molecular insights and mechanistic variations across sp ecies. Curr. Opin. Cell Biol. 17 316-325. Cougot,N., Babajko,S., and Seraphin,B. (2004). Cyt oplasmic foci are site s of mRNA decay in human cells. J. Cell Biol. 165, 31-40. Covey,S.N., Al-Kaff,N.S., Langara,A., and Turner,D.S. (1997). Plants combat infection by gene silencing. Nature 385, 781-782. Culjkovic,B., Topisirovic,I., Skrabanek,L., Ruiz-G utierrez,M., and Borden,K.L. (2006). eIF4E is a central node of an RNA regulon that governs cellular proliferation. J. Cell Biol. 175 415-426. Czech,B. et al. (2008). An endogenous small interferi ng RNA pathway in Drosophila. Nature 453, 798-802. Decker,C.J., Teixeira,D., and Parker,R. (2007). Edc3p and a glutamine/asparagine-rich domain of Lsm4p function in processing body assembly in Saccharomyces cerevisiae. J. Cell Biol. 179, 437-449. Ding,L., Spencer,A., Morita,K., and Han,M. (2005). The developmental timing regulator AIN-1 interacts with miRISCs and may target the argonaute protein AL G-1 to cytoplasmic P bodies in C. elegans. Mol. Cell 19 437-447. Durand,S., Cougot,N., Mahuteau-Betzer,F., Nguye n,C.H., Grierson,D.S., Bertrand,E., Tazi,J., and Lejeune,F. (2007). Inhibition of nonsense-mediated mRNA decay (NMD) by a new chemical molecule reveals the dynamic of NMD factors in Pbodies. J. Cell Biol. 178 11451160. El-Shami,M., Pontier,D., Lahmy,S., Braun,L., Pi cart,C., Vega,D., Hakimi,M.A., Jacobsen,S.E., Cooke,R., and Lagrange,T. (2007). Reiterate d WG/GW motifs form functionally and 101

PAGE 102

evolutionarily conserved ARGO NAUTE-binding platforms in R NAi-related components. Genes Dev. 21, 2539-2544. Era,T. and Witte,O.N. (2000). Regulated expres sion of P210 Bcr-Abl during embryonic stem cell differentiation stimulates multipotential progenito r expansion and myeloid cell fate. Proc. Natl. Acad. Sci. U. S. A 97, 1737-1742. Eulalio,A., Behm-Ansmant,I., and Izaurralde,E. (2007a). P bodies: at the crossroads of posttranscriptional pathways. Nat. Rev. Mol. Cell Biol. 8, 9-22. Eulalio,A., Behm-Ansmant,I., Schweizer,D., a nd Izaurralde,E. (2007b). P-body formation is a consequence, not the cause of RNA-medi ated gene silencing. Mol. Cell Biol. 27, 3970-3981. Eulalio,A., Huntzinger,E., and Izaurralde,E. (2 008). GW182 interacti on with Argonaute is essential for miRNA-mediated translational re pression and mRNA decay. Nat Struct. Mol. Biol. 15, 346-353. Eystathioy,T., Chan,E.K.L., Takeuchi,K., Mahler,M., Luft,L.M., Zochodne,D.W., and Fritzler,M.J. (2003a). Clinical a nd serological associations of autoantibodies to GW bodies and a novel cytoplasmic autoantigen GW182. J. Mol. Med. 81, 811-818. Eystathioy,T., Chan,E.K.L., Tenenbaum,S.A., Keene, J.D., Griffith,K., and Fritzler,M.J. (2002a). A phosphorylated cytoplasmic autoantigen, GW182, associates with a unique population of human mRNAs within novel cytoplasmic speckles. Mol. Biol. Cell 13, 1338-1351. Eystathioy,T., Jakymiw,A., Chan,E.K.L., Seraphi n,B., Cougot,N., and Fritzler,M.J. (2003b). The GW182 protein colocalizes with mRNA degradati on associated proteins hDcp1 and hLSm4 in cytoplasmic GW bodies. RNA. 9, 1171-1173. Eystathioy,T., Peebles,C.L., Hamel,J.C., Vaughn,J.H., and Chan,E.K.L. (2002b). Autoantibody to hLSm4 and the heptameric LSm comple x in anti-Sm sera. Arthritis Rheum. 46, 726-734. Famulski,J.K., Vos,L., Sun,X., and Chan,G. (2008). Stable hZW10 kinetochore residency, mediated by hZwint-1 interaction, is essent ial for the mitotic checkpoint. J. Cell Biol. 180 507520. Fenger-Gron,M., Fillman,C., Norrild,B., and Lykke-Andersen,J. (2005). Multiple processing body factors and the ARE binding protein TTP activate mRNA decapping. Mol. Cell 20 905915. Ferraiuolo,M.A., Basak,S., Dostie,J., Murray,E .L., Schoenberg,D.R., and Sonenberg,N. (2005). A role for the eIF4E-binding protein 4E-T in P-body formation and mRNA decay. J. Cell Biol. 170, 913-924. Filipowicz,W., Bhattacharyya,S.N., and S onenberg,N. (2008). Mechanisms of posttranscriptional regulation by microRNAs: are the answers in sight? Nat Rev. Genet. 9, 102-114. 102

PAGE 103

Filipowicz,W., Jaskiewicz,L., Kolb,F.A., and Pillai,R.S. (2005). Post-transcriptional gene silencing by siRNAs and miRNAs Curr. Opin. Struct. Biol. 15 331-341. Fire,A., Xu,S., Montgomery,M.K., Kostas,S.A., Driv er,S.E., and Mello,C.C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811. Franks,T.M. and Lykke-Andersen,J. (2007). TTP and BRF proteins nucleate processing body formation to silence mRNAs with AU-rich elements. Genes Dev. 21, 719-735. Gallois-Montbrun,S., Kramer,B., Swanson,C .M., Byers,H., Lynham,S., Ward,M., and Malim,M.H. (2007). Antiviral protein APOBEC3G localizes to ribonucl eoprotein complexes found in P bodies and stress granules. J. Virol. 81, 2165-2178. Gazzani,S., Lawrenson,T., Woodward,C., Headon,D., and Sablowski,R. (2004). A link between mRNA turnover and RNA interference in Arabidopsis. Science 306, 1046-1048. Ghildiyal,M. et al. (2008). Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells. Science 320 1077-1081. Griffith,J.L., Coleman,L.E., Raymond,A.S., Goodson,S.G., Pittard,W.S., Tsui,C., and Devine,S.E. (2003). Functional genomics reveals relationships between the retrovirus-like Ty1 element and its host Saccharomyces cerevisiae. Genetics 164, 867-879. Grimson,A., Farh,K.K., Johnston,W.K., Garrett-E ngele,P., Lim,L.P., and Bartel,D.P. (2007). MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol. Cell 27, 91-105. Hatfield,S.D., Shcherbata,H.R., Fischer,K.A., Nakahara,K., Carthew,R.W., and RuoholaBaker,H. (2005). Stem cell division is regulated by the microRNA pathway. Nature 435 974978. He,F. and Jacobson,A. (1995). Identification of a novel component of the nonsense-mediated mRNA decay pathway by use of an interacting protein screen. Genes Dev. 9, 437-454. He,L. et al. (2007a). A microRNA component of the p53 tumour suppressor network. Nature 447, 1130-1134. He,L., He,X., Lowe,S.W., and Hannon,G.J. (2007b). microRNAs join the p53 network--another piece in the tumour-suppression puzzle. Nat. Rev. Cancer 7, 819-822. He,L. et al. (2005). A microRNA polycistron as a potential human oncogene. Nature 435 828833. Heyer,W.D., Johnson,A.W., Reinhart,U., and Kolodner,R.D. (1995). Regulation and intracellular localization of Saccharomyces cerevisiae strand exchange pr otein 1 (Sep1/Xrn1/Kem1), a multifunctional exonuclease. Mol. Cell Biol. 15, 2728-2736. 103

PAGE 104

Ikawa,M., Kominami,K., Yoshimura,Y., Tanaka,K., Nishimune,Y., and Okabe,M. (1995). A rapid and non-invasive selection of transgen ic embryos before im plantation using green fluorescent protein (GFP). FEBS Lett. 375, 125-128. Ingelfinger,D., rndt-Jovin,D.J., Luhrmann,R., and Achsel,T. (2002) The human LSm1-7 proteins colocalize with the mRNA-degrading enzymes Dcp1/2 and Xrnl in distinct cytoplasmic foci. RNA. 8, 1489-1501. Irwin,B., Aye,M., Baldi,P., Be liakova-Bethell,N., Cheng,H., D ou,Y., Liou,W., and Sandmeyer,S. (2005). Retroviruses and yeast retr otransposons use overlapping sets of host genes. Genome Res. 15, 641-654. Jakymiw,A., Ikeda,K., Fritz ler,M.J., Reeves,W.H., Satoh,M., and Chan,E.K.L. (2006). Autoimmune targeting of key components of RNA interference. Arthritis Res. Ther. 8, R87. Jakymiw,A., Lian,S., Eystathioy,T., Li,S., Satoh,M., Hamel,J.C., Fritzler,M.J., and Chan,E.K.L. (2005). Disruption of GW bodies impairs ma mmalian RNA interference. Nat. Cell Biol. 7, 12671274. Jakymiw,A., Pauley,K.M., Li,S., Ikeda,K., Lia n,S., Eystathioy,T., Sat oh,M., Fritzler,M.J., and Chan,E.K.L. (2007). The role of GW/P-bodies in RNA processing and silencing. J. Cell Sci. 120, 1317-1323. Katahira,J., Miki,T., Takano,K., Maruhashi,M., Uchikawa,M., Tachibana,T., and Yoneda,Y. (2008). Nuclear RNA export factor 7 is locali zed in processing bodies and neuronal RNA granules through interacti ons with shuttling hnRNPs. Nucleic Acids Res. 36, 616-628. Kawamura,Y., Saito,K., Kin,T., Ono,Y., Asai,K., Sunohara,T., Okada,T.N., Siomi,M.C., and Siomi,H. (2008). Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells. Nature 453, 793-797. Kedersha,N., Stoecklin,G., Ayodele,M., Y acono,P., Lykke-Andersen,J., Fitzler,M.J., Scheuner,D., Kaufman,R.J., Golan,D.E., and Anders on,P. (2005). Stress granules and processing bodies are dynamically linked sites of mRNP remodeling. J. Cell Biol. 169 871-884. Kent,O.A. and Mendell,J.T. (2006). A small piec e in the cancer puzzle: microRNAs as tumor suppressors and oncogenes. Oncogene 25, 6188-6196. Lee,Y.S., Nakahara,K., Pham,J.W., Kim,K., He,Z., Sontheimer,E.J., and Carthew,R.W. (2004). Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA si lencing pathways. Cell 117, 69-81. Legault,P., Li,J., Mogridge,J., Kay,L.E., and Greenblatt,J. (1998). NM R structure of the bacteriophage lambda N peptide/boxB RNA complex: recognition of a GNRA fold by an arginine-rich motif. Cell 93, 289-299. Lejeune,F., Li,X., and Maquat,L.E. (2003). Nonsense-mediated mRNA decay in mammalian cells involves decapping, deadenylating, a nd exonucleolytic activities. Mol. Cell 12, 675-687. 104

PAGE 105

Lewis,B.P., Burge,C.B., and Bartel,D.P. (2005) Conserved seed pair ing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15-20. Lian,S., Fritzler,M.J., Katz,J., Hamazaki,T., Te rada,N., Satoh,M., and Chan,E.K.L. (2007). Small interfering RNA-mediated silencing induces targ et-dependent assembly of GW/P bodies. Mol. Biol. Cell 18 3375-3387. Lian,S., Jakymiw,A., Eystathi oy,T., Hamel,J.C., Fritzler,M.J. and Chan,E.K.L. (2006). GW bodies, microRNAs and the cell cycle. Cell Cycle 5, 242-245. Liu,J., Carmell,M.A., Rivas,F.V., Marsden,C.G., Thomson,J.M., Song,J.J., Hammond,S.M., Joshua-Tor,L., and Hannon,G.J. (2004). Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437-1441. Liu,J., Rivas,F.V., Wohlschlegel,J., Yates,J.R ., Parker,R., and Hannon,G.J. (2005a). A role for the P-body component GW182 in micr oRNA function. Nat. Cell Biol. 7, 1261-1266. Liu,J., Valencia-Sanchez,M.A., Hannon,G.J., and Parker,R. (2005b). MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies. Nat. Cell Biol. 7, 719-723. Livak,K.J. and Schmittgen,T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Del ta Delta C(T)) Method. Methods 25, 402-408. Lykke-Andersen,J. (2002). Identification of a hu man decapping complex associated with hUpf proteins in nonsense-mediated decay. Mol. Cell Biol. 22, 8114-8121. Lykke-Andersen,J. and Wagner,E. (2005). Recruitm ent and activation of mRNA decay enzymes by two ARE-mediated decay activation domains in the proteins TTP and BRF-1. Genes Dev. 19, 351-361. Meister,G., Landthaler,M., Patkaniowska,A., Do rsett,Y., Teng,G., and Tuschl,T. (2004). Human Argonaute2 mediates RNA cleavage target ed by miRNAs and siRNAs. Mol. Cell 15 185-197. Meister,G., Landthaler,M., Peters,L., Chen,P.Y ., Urlaub,H., Luhrmann,R., and Tuschl,T. (2005). Identification of novel argonaute-as sociated proteins. Curr. Biol. 15, 2149-2155. Meister,G. and Tuschl,T. (2004). Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343-349. Mello,C.C. and Conte,D., Jr. (2004). Reveali ng the world of RNA interference. Nature 431, 338342. Moser,J.J., Eystathioy,T., Chan,E.K., and Fritzle r,M.J. (2007). Markers of mRNA stabilization and degradation, and RNAi within astr ocytoma GW bodies. J. Neurosci. Res. 85, 3619-3631. Muhlrad,D. and Parker,R. (1994). Premature tr anslational termina tion triggers mRNA decapping. Nature 370, 578-581. 105

PAGE 106

Nilsen,T.W. (2007). Mechanisms of microRNA-mediated gene regula tion in animal cells. Trends Genet. 23, 243-249. Nilsen,T.W. (2008). Endo-siRNAs: yet another la yer of complexity in RNA silencing. Nat. Struct. Mol. Biol. 15, 546-548. O'Donnell,K.A., Wentzel,E.A., Zeller,K.I., Dang,C.V., and Mendell,J.T. (2005). c-Mycregulated microRNAs modulate E2F1 expression. Nature 435 839-843. Okamura,K., Balla,S., Martin,R., Liu,N., and La i,E.C. (2008a). Two distinct mechanisms generate endogenous siRNAs from bidirectional transcription in Drosophila melanogaster. Nat. Struct. Mol. Biol. 15, 581-590. Okamura,K., Chung,W.J., Ruby,J.G., Guo,H., Bart el,D.P., and Lai,E.C. (2008b). The Drosophila hairpin RNA pathway generates endoge nous short interfering RNAs. Nature 453, 803-806. Okamura,K., Ishizuka,A., Siomi,H., and Siom i,M.C. (2004). Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways. Genes Dev. 18, 1655-1666. Orban,T.I. and Izaurralde,E. (2005). Decay of mRNAs targeted by RISC requires XRN1, the Ski complex, and the exosome. RNA. 11 459-469. Parker,R. and Song,H. (2004). The enzymes and control of eukaryotic mRNA turnover. Nat. Struct. Mol. Biol. 11, 121-127. Pauley,K.M., Eystathioy,T., Jakymiw,A., Hamel,J.C., Fritzler,M.J., and Chan,E.K.L. (2006). Formation of GW bodies is a consequence of microRNA genesis. EMBO Rep. 7, 904-910. Pauley,K.M., Satoh,M., Dominguez-Gutierrez,P.R., Pop,S.M., Holliday,S.L., Reeves,W.H., and Chan,E.K.L. (2008). Formation of GW/P bodies as marker for microRNA-mediated regulation of innate immune signaling. Mo l. Biol. Cell (in revision). Pillai,R.S., Artus,C.G., and Filipowicz,W. (2004). Tethering of human A go proteins to mRNA mimics the miRNA-mediated repre ssion of protein synthesis. RNA 10, 1518-1525. Pillai,R.S., Bhattacharyya,S.N., Artus,C.G., Zoller,T., Cougot,N., Basyuk,E., Bertrand,E., and Filipowicz,W. (2005). Inhibition of translational initiation by Let-7 MicroRNA in human cells. Science 309, 1573-1576. Ratcliff,F., Harrison,B.D., and Ba ulcombe,D.C. (1997). A similar ity between viral defense and gene silencing in plants. Science 276 1558-1560. Rehwinkel,J., Behm-Ansmant,I., Gatfield,D., and Izaurralde,E. (2005). A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing. RNA. 11, 16401647. 106

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Schneider,M.D., Najand,N., Chaker,S., Pare,J.M ., Haskins,J., Hughes,S.C., Hobman,T.C., Locke,J., and Simmonds,A.J. (2006). Gawky is a component of cytoplasmic mRNA processing bodies required for early Drosoph ila development. J. Cell Biol. 174, 349-358. Sen,G.L. and Blau,H.M. (2005). Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies. Nat. Cell Biol. 7, 633-636. Serman,A., Le,R.F., Aigueperse,C., Kress,M ., Dautry,F., and Weil,D. (2007). GW body disassembly triggered by siRNAs independently of their silencing activity. Nucleic Acids Res. 35, 4715-4727. Sheth,U. and Parker,R. (2003). Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science 300, 805-808. Souret,F.F., Kastenmayer,J.P., and Green,P.J. (2004). AtXRN4 degrades mRNA in Arabidopsis and its substrates include selected miRNA targets. Mol. Cell 15 173-183. Tahbaz,N., Kolb,F.A., Zhang,H., Jaronczyk,K ., Filipowicz,W., and Hobman,T.C. (2004). Characterization of the interactions between ma mmalian PAZ PIWI domain proteins and Dicer. EMBO Rep. 5, 189-194. Tam,O.H. et al. (2008). Pseudogene-derived small interfer ing RNAs regulate ge ne expression in mouse oocytes. Nature 453 534-538. Teixeira,D. and Parker,R. (2007). Analysis of P-Body Assembly in Saccharomyces cerevisiae. Mol. Biol. Cell 18, 2274-2287. Teixeira,D., Sheth,U., Valencia-Sanchez,M.A., Brengues,M., and Parker,R. (2005). Processing bodies require RNA for assembly and contain nontranslating mRNAs. RNA. 11, 371-382. Till,S., Lejeune,E., Thermann,R., Bortfeld,M., Hothorn,M., Enderle,D., Heinrich,C., Hentze,M.W., and Ladurner,A.G. (2007). A conser ved motif in Argonaute-interacting proteins mediates functional interactions through the Argonaute PIWI do main. Nat Struct. Mol. Biol. 14, 897-903. Tourriere,H., Chebli,K., and Tazi,J. (2002). mRNA degradation machines in eukaryotic cells. Biochimie 84, 821-837. van Dijk,E., Cougot,N., Meyer,S., Babajko,S., Wa hle,E., and Seraphin,B. (2002). Human Dcp2: a catalytically active mRNA decapping enzyme lo cated in specific cytoplasmic structures. EMBO J. 21 6915-6924. Vasudevan,S., Tong,Y., and Steitz,J.A. (2007). Switching from repression to activation: microRNAs can up-regulate translation. Science 318, 1931-1934. Watanabe,T. et al. (2008). Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453, 539-543. 107

PAGE 108

108 Wedekind,J.E., Dance,G.S., Sowden,M.P., and Smith,H.C. (2003). Messenger RNA editing in mammals: new members of the APOBEC family s eeking roles in the family business. Trends Genet. 19, 207-216. Wichroski,M.J., Robb,G.B., and Rana,T.M. (2006). Human retroviral host restriction factors APOBEC3G and APOBEC3F localize to mRNA processing bodies. PLoS. Pathog. 2, e41. Wilusz,C.J., Wormington,M., and Peltz,S.W. (2001). The cap-to-tail guide to mRNA turnover. Nat. Rev. Mol. Cell Biol. 2, 237-246. Yang,W.H., Yu,J.H., Gulick,T., Bloch,K.D., and Bloch,D.B. (2006). RNA-a ssociated protein 55 (RAP55) localizes to mRNA processing bodies and stress granules. RNA. 12, 547-554. Yang,Z., Jakymiw,A., Wood,M.R., Eystathioy,T., Rubin,R.L., Fritzler,M.J., and Chan,E.K.L. (2004). GW182 is critical for the stability of GW bodies expresse d during the cell cycle and cell proliferation. J. Cell Sci. 117, 5567-5578. Yedavalli,V.S., Neuveut,C., Chi,Y.H., Kleiman,L ., and Jeang,K.T. (2004). Requirement of DDX3 DEAD box RNA helicase for HI V-1 Rev-RRE export function. Cell 119, 381-392. Yu,D. et al. (2007). Roquin represses autoimmunity by limiting inducible T-cell co-stimulator messenger RNA. Nature 450, 299-303. Yu,J.H., Yang,W.H., Gulick,T., Bloch,K.D., and Bloc h,D.B. (2005). Ge-1 is a central component of the mammalian cytoplasmic mRNA processing body. RNA. 11, 1795-1802. Yuan,Y.R., Pei,Y., Ma,J.B., Kuryavyi,V., Zhadina,M., Meister,G., Chen,H.Y., Dauter,Z., Tuschl,T., and Patel,D.J. (2005). Crystal structur e of A. aeolicus argonaute, a site-specific DNAguided endoribonuclease, provide s insights into RISC-mediate d mRNA cleavage. Mol. Cell 19, 405-419.

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BIOGRAPHICAL SKETCH Shang Li Lian was born on Dec 7, 1977, in Guangzhou, China. She studied clinical medicine at Sun Yat-sen Universi ty, a famous medical school in Ch ina, and received Bachelor of Medicine in 2002. Inspired by her father, Shang got interested in biomedical research which helped her further understand medical science from a brand new angle. She was admitted and entered the Interdisciplinary Program in Biomedi cal Sciences in College of Medicine at the University of Florida in 2003. Shang joined th e laboratory of Dr. Edward K.L. Chan in the summer of 2004. She began her research on the function of GW182 and GW/P body in RNA interference. She received Ph.D. degree in Medical Sciences-Molecula r Cell Biology in Aug 2008. Shang planned to continue her research on the cell biology of RNA interference. Her ultimate goal is to become a pathologist and eventually start her own laboratory. During her Ph.D. study, Shang was married to Songqing Li in Oct 2004. 109