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Novel Functions of Smn Complex Members and Their NOVEL FUNCTIONS OF SMN COMPLEX MEMBERS AND THEIR IMPLICATIONS IN SPINAL MUSCULAR ATROPHY By MICHAEL PATRICK WALKER 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 May, 2009 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 tables...........................................................................................................5 List of figures…………………………………………………………………….6 Acknowledgements……………………………………………………………...8 Abbreviations…………………………………………………………….............9 Abstract……………………………………………………………………………10 Chapter I: Introduction and Research Objectives…………………………...12 Introduction…………………………………………………………13 Spinal Muscular Atrophy……………………………………13 snRNP Biogenesis…………………………………….............21 The SMN Complex…………………………………………...26 SMN and Gemin Function in the Motor Unit……………...33 Research Objectives…………………………………………………39 Chapter II: Overexpression of Gemin4 relocalizes the SMN complex to the nucleoplasm and causes disassembly of Cajal Bodies…………………..41 Abstract…………………………………………………………….....42 Introduction…………………………………………………..............43 Results…………………………………………………………………46 Discussion……………………………………………………………..59 Materials and Methods………………………………………………61 3 Chapter III: Characterization of Gemin4 loss-of-function in Mus musculus.63 Abstract……………………………………………………………........64 Introduction………………………………………………….................65 Results…………………………………………………………………...69 Discussion………………………………………………………….........75 Materials and Methods………………………………………………...77 Chapter IV: The SMN complex localizes to the sarcomeric Z-disc and is a proteolytic target of calpain………………………………………………………..79 Abstract……………………………………………………………........80 Introduction………………………………………………….................81 Results…………………………………………………………………...85 Discussion……………………………………………………………….104 Material and Methods…………………………………………………109 Chapter V: Discussion and Future Directions………………………..................113 Implications of Gemin4 Mediated Import………………..................114 Gemin4 Mice and SMA……………………………………..................119 A Novel Function of the SMN Complex in Striated Muscle………123 Appendix Chapter: Reduced viability and fecundity in mice lacking the Cajal body marker protein, coilin………………………………………………...131 Abstract…………………………………………………………………132 Introduction……………………………………………………….........133 Results………………………………………………………...................136 Discussion……………………………………………………………….144 Material and Methods…………………………………………………149 Bibliography…………………………………………………………………………150 4 List of Tables Chapter III Table 3-1: Gemin4 is essential in the mouse…………………………....71 Table 3-2: Genetic background contribution to the SMA phenotype..72 Table 3-3: Gemin4 heterozygotes do not modify the SMA early lethality phenotype……………………………………...........73 Appendex Chapter Table A1-1: Coilin intercross mice……………………………………….137 Table A1-2: Contibution of the uterine environment to neonatal viability………………………………………………………138 5 List of Figures Chapter I Figure 1-1: SMN2 exon 7 skipping………………………………………….15 Figure 1-2: SMN2 expression inversely correlates with the SMA phenotype………………………………………………..............16 Figure 1-3: A simplified overview of mRNA splicing……………............22 Figure 1-4: snRNP biogenesis overview………………………...................25 Figure 1-5: The SMN complex………………………………………..............27 Figure 1-6: Functions of SMN & Gemins in the motor unit…..................34 Figure 1-7: The Sarcomere…………………………………………………..38 Chapter II Figure 2-1: GFP:Gemin4 localizes to the nucleus…………………………47 Figure 2-2: GFP:Gemin4 deletion constructs……………………………...48 Figure 2-3: The amino terminal half of Gemin4 contains a nuclear localization signal…………………………………….…………49 Figure 2-4: The Gemin4 NLS is sufficient to drive nuclear import……..50 Figure 2-5: Mouse and human Gemin4 localize to the nucleus Regardless of the placement of GFP…………………………51 Figure 2-6: Gemin4 redirects Gemin3 into the nucleus……………..........53 Figure 2-7: Gemin4 can redirect other members of the SMN complex into the nucleus………………………………………………...56 Figure 2-8: Cajal Bodies are maintained in GFP:Gemin4 expressing Cells……………………………………………………………….58 6 Chapter III Figure 3-1: Characterization of Gemin4 gene trap………………………..70 Figure 3-2: Gemin4 expression profile…………………………………….74 Figure 3-3: Gemin4 heterozygous expression profile…………………...74 Chapter IV Figure 4-1: Smn localizes to skeletal myofibrils………………….............86 Figure 4-2: Smn localizes to the Z-discs of striated muscle……………..87 Figure 4-3: The Smn complex is present at Z-discs………………………89 Figure 4-4: U snRNPs do not localize to myofibrils……………………..90 Figure 4-5: Calpain activity removes Smn from myofibrils…………….92 Figure 4-6: Calpain directly cleaves SMN in vitro………………………..94 Figure 4-7: Characterization of novel anti-SMN monoclonal antibody 9F2……………………………………………………………….95 Figure 4-8: Calpain inhibition, titration and endogenous activation…..97 Figure 4-9: Inhibition of calpain cleavage in human and mouse cell lysates using a peptide inhibitor………………………………98 Figure 4-10: Calpain cleaves a subset of the Gemins within the SMN complex in vitro and in vivo……………………………...…....100 Figure 4-11: SMA type 1 mice have aberrant myofibrils……………….103 Chapter V Figure 5-1: The Gemin4 Import Receptor………………………..............116 Figure 5-2: Gemin4 Mediated Import……………………………….........121 Figure 5-3: Schematic of Gemin4 Embryo Analysis ……………….........123 Figure 5-4: Is RNP biogenesis occurring at the Z-disc?.....……………...125 Figure 5-5: Possible functions of the SMN complex at the Z-disc……..127 7 Figure 5-6: mRNP transport for maintaining myofibrils……………….131 Appendix Chapter Figure A1-1: Coil -/- mice are reproductively less fit…………………..140 Figure A1-2: Reciprocal mating crosses…………………………………142 Figure A1-4: Coil -/- mice have reduced testis size…………….………..144 Figure A1-5: Analysis of Coil -/- ovaries…………………………………145 8 Acknowledgements I would like to take this opportunity to thank my mentor, Dr. Gregory Matera. Greg is an extraordinarily bright scientist and over the years it has been a privilege to interact and learn from him. Greg has always been there for discussions of my project, science, and life in general. He has always been able to point out the “big picture”, when, at times I was discouraged about the way my research was heading. Greg really knew when to let me be independent and when to step in and get me back on the right track, for that I’ll always be truly grateful. I would also like to thank all the members of the Matera lab, past and present. Not only were they great colleagues whose knowledge and suggestions played an integral role in my development as a scientist, but I also consider all of them good friends for the rest of my life. Thank you to my committee for their advice and support over the years with regard to my project. I would like to thank my parents, grandparents and siblings for their unwavering support throughout my graduate career and giving me the courage to follow my dreams. Most importantly, I would like to thank my wife, Heather. She has been a constant source of inspiration, support and companionship through out all of this and I know her being in my life has given me the drive to succeed; for it was here in graduate school she made the happiest man alive and married me and gave me the proudest day in my life, the birth of our daughter, Blair. 9 List of abbreviations Aa amino acid ATP adenosine triphospate CBC cap binding complex GFP green fluorescent protein GIP1 Gemin3 interaction protein 1 GST glutathione S-transferase hnRNP heterogeneous nuclear RNP IP immunoprecipitation KO knock out mRNA messenger RNA NLS nuclear localization signal NMJ neuromuscular junction NPC nuclear pore complex PRMT5 protein arginine methyltranferase 5 PCR polymerase chain reaction RNP ribonucleoprotein RNA ribose nucleic acid SIP1,2,3 SMN interacting protein 1,2,3 SMA spinal muscular atrophy SMN survival of motor neuron snoRNP small nucleolar RNP snRNA small nuclear RNA snRNP small nuclear RNP SPN snurportin TMG 2,2,7-trimethylguanosine U snRNP uridine-rich snRNP UNRIP unr interacting protein 10 Novel Functions of SMN Complex Members and Their Implications in Spinal Muscular Atrophy Abstract By MICHAEL PATRICK WALKER Spinal Muscular Atrophy (SMA) is one of the most widespread genetic disorders in children. Genetic research into the disorder has identified the survival motor neurons 1 (SMN1) gene as the causative agent. The gene product, SMN, is found in a multiprotein particle, the SMN complex, required for the biogenesis of essential splicing factors, the U snRNPs. While much is known about the function of SMN within this complex, other member’s roles remain enigmatic. Recent investigations in muscle and motoneurons also point to additional functions for the SMN complex outside of snRNP biogenesis. These revelations have lead to a debate on whether or not SMA results from faulty snRNP production or from perturbation of another function(s). Clearly, a better understanding
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