GEMIN FUNCTION IN SMALL NUCLEAR RNP BIOGENESIS AND SPINAL MUSCULAR ATROPHY By KARL BRYAN SHPARGEL Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisor: Dr. A. Gregory Matera Department of Genetics CASE WESTERN RESERVE UNIVERSITY August, 2006 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents List of figures………………………………………………………………………….....6 Acknowledgements………………………………………………………………......…..8 Abbreviations………………………………………………………………………..…...9 Abstract………………………………………………………………………………….10 Chapter I: Introduction and Research Objectives………………………...…………12 Introduction………………………………………………………………………13 Spinal Muscular Atrophy…………………………………………...……13 The SMN complex……………………………………………………….16 snRNP biogenesis……………………………………………………..…18 The Cajal body…………………………………………………………...22 Dilemmas in SMA pathogenesis and snRNP biogenesis…………...……26 Research Objectives……………………………………………………...………29 Chapter II: Gemin proteins are required for efficient assembly of Sm-class RNPs………………………………………………………….30 Abstract………………………………………………………………………..…31 Introduction………………………………………………………………………32 Materials and Methods…………………………………………………………...35 Results and Discussion…………………………………………………………..38 3 Chapter III: Drosophila Gemin3 is essential for larval motor function, pupation, and viability………………………………….……56 Abstract…………………………………………………………………………..57 Introduction…………………………………………………………………...….58 Materials and Methods………………………………………………………...…62 Results………………………………………………………………………...….65 Discussion……………………………………………………………..…………85 Chapter IV: Discussion and future directions………………………………..………88 What factors regulate Cajal body homeostasis?....................................................89 Gemin proteins function in snRNP assembly, but by what mechanism?............................................................................................................91 Are Gemins required for additional snRNP biogenesis steps?..............................94 Can Gemin snRNP biogenesis defects promote Spinal Muscular Atrophy?................................................................................................95 Which SMN functions are required for appropriate neuromuscular development?................................................................................97 Does the primary SMA defect originate in neurons or muscle (or both)? ...............................................................................................................98 Why does snRNP biogenesis have such a profound effect on motor neurons?.....................................................................................................103 Appendix Chapter I: Control of Cajal body number is mediated by the coilin C-terminus……………………………………...108 Abstract…………………………………………………………………...….…109 Introduction……………………………………………………………….....….110 Materials and Methods……………………………………………………...…..114 Results…………………………………………………………………………..115 Discussion……………………………………………………………………....134 4 Appendix Chapter II: Exogenous Gemin4 expression enhances Gemin3 and SMN nuclear localization…………….……….138 Abstract…………………………………………………………...…………….139 Materials and Methods…………………………………….……………………140 Results and Discussion…………………………………………………………141 Bibliography…………………………………………………..……………………….147 5 List of Figures Chapter I Figure 1-1: Mutations in SMN1 result in SMA…………………………………..14 Figure 1-2: SMN forms a large macromolecular complex that interacts with Sm proteins………………………………………17 Figure 1-3: SMN protein domain structure………………………………………17 Figure 1-4: snRNP biogenesis overview…………………………………………21 Figure 1-5: The Cajal body functions in snRNP, snoRNP, and teRNP maturation and assembly………………………………..25 Figure 1-6: SMN is involved in several cellular functions………………………28 Chapter II Figure 2-1: SMN and Gemin protein levels are interdependent…………..……..39 Figure 2-2: SMN, Gemin2, Gemin3, and Gemin4 are required for efficient Sm core assembly…………………………………..….41 Figure 2-3: Loss of SMN and snurportin results in breakdown of nuclear Cajal bodies………………………………………………44 Figure 2-4: SMN and Gemins regulate Sm core assembly in vivo………………48 Figure 2-5: SMA patient-derived SMN mutations are defective in Sm core assembly……………………………………………..…52 Figure 2-6: Supplemental figure…………………………………………………54 Chapter III Figure 3-1: Drosophila Gemin3 is conserved throughout helicase motifs..…..…66 Figure 3-2: dGemin3 interacts with dSMN in vitro and in vivo…………………68 Figure 3-3: Drosophila SMN and Gemin3 are required for efficient snRNA Sm core assembly…………………………...…….70 Figure 3-4: Smn and Gemin3 mutant larvae exhibit viability and pupation defects………………………………………...………74 6 Figure 3-5: Smn and Gemin3 mutants exhibit growth defects……………..…….77 Figure 3-6: Smn and Gemin3 mutant larvae exhibit defects in motor function…………………………………………………….79 Figure 3-7: Smn and Gemin3 mutants exhibit neuronal pathfinding defects…………………………………………..………80 Figure 3-8: Smnex33, SmD2, and Gemin3 interact genetically……………...…….83 Chapter IV Figure 4-1: Models for SMN complex function in snRNP Sm core assembly……………………………………..……...……..94 Figure 4-2: Experimental analysis of tissue specificity in SMA………….……103 Figure 4-3: Models for tissue specific SMA pathogenesis………………..……107 Chapter AI Figure A1-1: Deletion of the coilin self-association domain affects epitope recognition………………………………….……116 Figure A1-2: Coilin mutants display unregulated nuclear body formation…………………………………………………...119 Figure A1-3: Coilins from different species display variation in nuclear body formation…………………………………….….122 Figure A1-4: Schematic of human and mouse coilin………………...…………123 Figure A1-5: Mutations in mouse coilin affect nuclear body formation……….127 Figure A1-6: Ectopically expressed SMN produces numerous SMN foci……..132 Chapter AII Figure A2-1: Gemin3 overexpression localizes SMN to the Golgi complex…..142 Figure A2-2: Golgi dissociation alters Gemin3 localization………………...…143 Figure A2-3: Gemin4 overexpression imports Gemin3 and SMN into the nucleus……………………………...………..……145 7 Acknowledgements I would like to take this opportunity to thank my research advisor, Greg Matera. Greg’s enthusiastic approach to research has left a very positive impression on me. There were times when I doubted whether I would ever be able to finish my project, but Greg was always convinced that the end was somewhere within reach. He has been a great mentor and I will always cherish our discussions about research, life, or football. I also would like to thank all members of the Matera Lab who have been instrumental in my training as well as helping me keep my sanity. Thank you to my thesis committee for their advise in the direction of my project. I would like to thank my parents, Jerold and Shirley, and sisters, Sara and Rebecca, for their unending support and inspiration to follow my dreams in life. Finally and perhaps most importantly, I would like to thank my wife, Tarah. She has put up with all the late worknights, weekend trips to the lab, and life with a graduate student stipend. Throughout the tumultuous graduate school life, she has been my smile at the end of the day and continued to give me confidence that this graduation day will come. And a special thanks to her for giving me my son Jack, a fantastic graduation gift! 8 List of abbreviations ATP Adenosine triphosphate CB Cajal body CBC Cap binding complex DAPI 4′,6-diamidino-2-phenylindole DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid Gem3 Gemin3 GFP Green fluorescent protein GST Glutathione S-transferase GTP Guanosine triphosphate His Histidine IP Immunoprecipitation IPTG Isopropyl-beta-D-Thiogalactopyranoside m7G 7-methylguanosine mRIPA Modified radioimmunoprecipitaion assay myc c-Myc epitope NLS Nuclear localization signal NP-40 Nonidet-40 NPC Nuclear pore complex PBS Phosphate buffered saline PCR Polymerase chain reaction PHAX Phosphorylated adaptor for snRNA export PRMT5 Protein arginine methyltransferase rRNA ribosomal RNA RNA Ribonucleic acid RNP Ribonucleoprotein SMA Spinal Muscular Atrophy SMN Survival of Motor Neurons scaRNP small Cajal body specific RNP snRNA small nuclear RNA snRNP small nuclear RNP snoRNP small nucleolar RNP SPN snurportin teRNP telomerase RNP Tgs1 trimethylguanosine synthase TMG 2,2,7-trimethylguanosine Unrip unr interacting protein WT Wildtype 9 Gemin function in small nuclear RNP biogenesis and Spinal Muscular Atrophy Abstract By KARL BRYAN SHPARGEL The heart of genetics research lies in gaining knowledge and understanding of the effects that gene alterations have on human health and development. Ultimately, a thorough analysis of genetic diseases spanning from mutation identification to protein function will hopefully lead to the development of therapeutic treatments. Spinal Muscular Atrophy (SMA) is one of the most prevalent and dire genetic diseases. Hallmarks of the disease include degeneration of spinal motor neurons and skeletal muscular atrophy. The disease is particularly crippling