University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2011-04-05 Molecular Mechanisms of piRNA Biogenesis and Function in Drosophila: A Dissertation Chengjian Li University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Amino Acids, Peptides, and Proteins Commons, Animal Experimentation and Research Commons, Biochemistry, Biophysics, and Structural Biology Commons, Cells Commons, Genetic Phenomena Commons, and the Nucleic Acids, Nucleotides, and Nucleosides Commons Repository Citation Li C. (2011). Molecular Mechanisms of piRNA Biogenesis and Function in Drosophila: A Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/wq49-bk19. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/524 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. MOLECULAR MECHANISMS OF PIRNA BIOGENESIS AND FUNCTION IN DROSOPHILA A Dissertation Presented By CHENGJIAN LI Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY APRIL 5, 2011 BIOCHEMISTRY AND MOLECULAR PHARMACOLOGY iv COPYRIGHT INFORMATION The chapters of this dissertation have appeared in whole or part in publications below: Vagin, V. V.*, Sigova, A.*, Li, C., Seitz, H., Gvozdev, V., and Zamore, P. D. (2006). A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313, 320-324. Horwich, M. D.*, Li, C.*, Matranga, C.*, Vagin, V., Farley, G., Wang, P., and Zamore, P. D. (2007). The Drosophila RNA methyltransferase, DmHen1, modifies germline piRNAs and single-stranded siRNAs in RISC. Curr Biol 17, 1265-1272. Li, C.*, Vagin, V. V.*, Lee, S.*, Xu, J.*, Ma, S., Xi, H., Seitz, H., Horwich, M. D., Syrzycka, M., Honda, B. M., Kittler, E. L., Zapp, M. L., Klattenhoff, C., Schulz, N., Theurkauf, W. E., Weng, Z., and Zamore, P. D. (2009). Collapse of germline piRNAs in the absence of Argonaute3 reveals somatic piRNAs in flies. Cell 137, 509-521. * These authors contributed equally to this work. v DEDICATION To my parents, my brothers, my wife, and my son with love vi ACKNOWLEDGMENTS First of all, I would like to thank my advisor, Phillip Zamore, for giving me the opportunity to work in his lab, for his mentoring and confidence in my research, and for his support to my family. I feel extremely lucky to have worked with Phil. His enthusiasm and attitudes for science as well as life exert invaluable influence on me. I deeply appreciate every minute he has spent with me. I would also like to thank all the alumni (Alla, Ben, Brad, Chris, Dianne, Hervè, Irena, Klaus, Megha, Mike, Shengmei, Tingting, Vasia, Yonatan, and Yuki), and current members (Alicia, Bo, Cindy, Desi, Elif, Fabian, Gwen, Jen, Jogi, Keith, Ryuya, Stefan, Tiffanie, Tim, Tracey, Wee, Xin, and Zhao) in Phil’s lab for their friendship, for their efforts in creating such a great scientific environment, for the joyful time in the past few years. I want to express my gratitude to Bill Theurkauf and his Lab (particularly Nadine and Carla) as well as Zhiping Weng and her Lab (particularly Jia, Soo, Jie and Jui-Hung) for the wonderful collaborations. I would also like to acknowledge the members of my committee, Melissa Moore, Sean Ryder, Bill Theurkauf, and Scot Wolfe, for their insightful comments and suggestions on my research as well as on my career. I would also like to thank Gregory Hannon for being the external member of my committee. I want to extend my appreciation to people in UMASS Medical School and University of Pittsburgh for their support, friendship, and advice, especially, Marc Freeman, Guangping Gao, Bill Kobertz, Oliver Rando, and Michael Tsang. For all of their support and help, I would like to thank my friends, especially, Daorong Guo, Kai Li, Randong Shan, Guoming Sun, and Lei Wang. vii Most importantly, I would like to thank my parents and my brother’s families for their tremendous love and support despite the geographic distance. Most of all, I want to thank my wife, Xiaolan, for her constant love and support, as well as bringing our son, Alan, to the world, who really helped me learn how to be a good father, and at the same time deeply understand the love from my parents. viii ABSTRACT In the Drosophila germ line, PIWI-interacting RNAs (piRNAs) ensure genomic stability by silencing endogenous selfish genetic elements such as retrotransposons and repetitive sequences. We examined the genetic requirements for the biogenesis and function of piRNAs in both female and male germ line. We found that piRNAs function through the PIWI, rather than the AGO, family Argonaute proteins, and the production of piRNAs requires neither microRNA (miRNA) nor small interfering RNA (siRNA) pathway machinery. These findings allowed the discovery of the third conserved small RNA silencing pathway, which is distinct from both the miRNA and RNAi pathways in its mechanisms of biogenesis and function. We also found piRNAs in flies are modified. We determined that the chemical structure of the 3´-terminal modification is a 2´-O-methyl group, and also demonstrated that the same modification occurs on the 3´ termini of siRNAs in flies. Furthermore, we identified the RNA methyltransferase Drosophila Hen1, which catalyzes 2´-O-methylation on both siRNAs and piRNAs. Our data suggest that 2´-O-methylation by Hen1 is the final step of biogenesis of both the siRNA pathway and piRNA pathway. Studies from the Hannon Lab and the Siomi Lab suggest a ping-pong amplification loop for piRNA biogenesis and function in the Drosophila germline. In this model, an antisense piRNA, bound to Aubergine or Piwi, triggers production of a sense piRNA bound to the PIWI protein Argonaute3 (Ago3). In turn, the new piRNA is envisioned to produce a second antisense piRNA. We isolated the loss-of-function mutations in ago3, allowing a direct genetic test of ix this model. We found that Ago3 acts to amplify piRNA pools and to enforce on them an antisense bias, increasing the number of piRNAs that can act to silence transposons. Moreover, we also discovered a second Ago3-independent piRNA pathway in somatic ovarian follicle cells, suggesting a role for piRNAs beyond the germ line. x TABLE OF CONTENTS TITLE ii SIGNATURES iii COPYRIGHT INFORMATION iv DEDICATION v ACKNOWLEDGEMENTS vi ABSTRACT viii TABLE OF CONTENTS x LIST OF FIGURES xvi LIST OF TABLES xx CHAPTER I: INTRODUCTION 1 The core of small RNA pathways 2 Argonaute proteins 2 The discovery of piRNAs 6 The biogenesis of piRNAs does not require Dicer 6 The 3ˊ terminal modification is the last step of the biogenesis of piRNAs 7 The piRNA clusters are the source of piRNAs 10 The secondary piRNAs are generated by ping-pong amplification 12 A hypothetical model for the biogenesis of primary piRNAs 15 Nuage is a cellular compartment for piRNA production 17 Methylated arginines are the molecular linkers of the piRNA pathway 18 Drosophila piRNAs are born to combat genomic parasites 24 xi The regulation of protein-coding genes by piRNAs 25 CHAPTER II: A DISTINCT SMALL RNA PATHWAY SILENCES SELFISH GENETIC ELEMENTS IN THE GERM LINE 27 Preface 28 Summary 29 Results and Discussion 30 Phased sense and antisense siRNAs in vivo 30 Su(Ste) rasiRNAs 33 A third RNA silencing pathway in flies 39 Are roo rasiRNAs not made by dicing? 43 rasiRNAs bind Piwi and Aub 49 Materials and Methods 52 Fly stocks 52 Tiling microarrays 52 Microarray data analysis 53 RNA isolation and detection by Northern blot 54 Generation of mutant germ line clones 55 Analysis of rasiRNA and miRNA chemical structure 56 Quantitative RT-PCR analysis 57 Immunoprecipitation 58 Western blotting 59 Acknowledgments 61 CHAPTER III: THE DROSOPHILA RNA METHYLTRANSFERASE, DMHEN1, MODIFIES BOTH PIRNAS AND SINGLE-STRANDED SIRNAS IN RISC AND IS xii REQUIRED FOR SILENCING SELFISH GENETIC ELEMENTS IN THE GERM LINE 78 Preface 79 Summary 80 Results and Discussion 82 Drosophila piRNAs are 2´-O-methylated at their 3´ termini 82 Drosophila siRNAs are 2´-O-methylated at their 3´ termini 82 DmHen1 is required for piRNA modification in vivo 85 DmHen1 is required for piRNA function in vivo 88 DmHen1 is required for siRNA modification 91 siRNA modification correlates with Ago2-RISC assembly in vitro 94 siRNAs are modified only after Ago2-RISC maturation 96 Recombinant DmHen1 modifies single-stranded small RNA 99 Experimental Procedures 101 General Methods 101 32P-radiolabeled 3´ mononucleotide standards 101 2D-TLC 101 Analysis of RNA 3´ termini 102 Recombinant Drosophila Hen1 Protein 103 Analysis of double- and single-stranded siRNA 103 Acknowledgments 105 CHAPTER IV: COLLAPSE OF GERM-LINE PIRNAS IN THE ABSENCE OF ARGONAUTE3 REVEALS SOMATIC PIRNAS IN FLIES 122 Preface 123 xiii Summary 124 Introduction 125 Results 128 Loss-of-function ago3 alleles 128 Mutually interdependent localization of PIWI proteins 128 ago3 mutations affect fecundity 134 Silencing selfish genetic elements in germ line requires Ago3 137 Genome-wide piRNA analysis 139 Ago3 limits sense piRNA accumulation and amplifies antisense piRNAs 140 Three piRNA groups 141 Group I transposons require Ago3 for antisense piRNA amplification 144 Group II transposons act