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Investigation of RNA Quality Control Pathways in RNP Hypo-Assembly

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Investigation of RNA quality control pathways in RNP hypo-assembly diseases by Siddharth Shukla Integrated M.Sc., Indian Institute of Technology Bombay, 2011 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirement for the degree of Doctor of Philosophy Department of Chemistry and Biochemistry 2016 i This thesis entitled: Investigation of RNA quality control pathways in RNP hypo-assembly diseases written by Siddharth Shukla has been approved for the Department of Chemistry and Biochemistry ________________________________ Roy Parker ________________________________ James Goodrich Date ___________ The final copy of this thesis has been examined by the signatories, and we find that both the content and the form meet acceptable presentation standards of scholarly work in the above mentioned discipline. ii Shukla, Siddharth (Ph.D., Biochemistry) Investigation of RNA quality control pathways in RNP hypo-assembly diseases Thesis directed by Professor Roy Parker A key aspect of cellular function is the proper assembly and utilization of ribonucleoproteins (RNPs). Defects in the formation of RNPs lead to "RNP hypo- assembly diseases", which can be caused by RNA degradation out-competing RNP assembly. Examples of such human diseases include Dyskeratosis Congenita (DC) and Spinal Muscular Atrophy (SMA). In order to test the hypothesis that specific RNA quality control pathways were responsible for degradation of RNAs in these diseases, I used yeast and mammalian cell lines as model systems to investigate two different diseases, SMA and DC. In SMA, Sm-site mutations in yeast U1 snRNA led to their rapid degradation by two different RNA decay pathways: 3’ to 5’ decay in the nucleus by Trf4/Rrp6, and 5’ to 3’ decay in the cytoplasm by Dcp2/Xrn1. Cytoplasmic degradation of snRNAs when the Sm site is mutated is conserved in human cells. The cytoplasmic decay pathway is also responsible for the degradation of snRNAs when SMN levels are reduced, as is the case in SMA. Importantly, inhibition of snRNA decapping through DCP2 knockdown rescued splicing defects observed for some mRNAs when SMN is limiting. These results suggest that inhibition of snRNA decay could rescue some phenotypes of SMA. For the investigation of human telomerase RNA (hTR) quality control pathways in DC, disease causing mutations were introduced in hTR, along with the depletion of dyskerin in human cells. These models established that hTR is degraded by PAPD5/EXOSC10 in the nucleus, and DCP2/XRN1 in the cytoplasm. Inhibition of hTR decay rescued both the sub-cellular localization of hTR, as well as telomerase activity in iii human cells. Additionally, PARN, which is a 3’ to 5’ exonuclease, stabilized hTR through deadenylation of its 3’ end. PARN mutations also lead to a severe form of DC. I have identified other possible substrates of PARN in human cells, which suggest that PARN deadenylates a number of stable non coding RNAs in human cells, but influences the stability of a select few. This potentially explains why PARN mutations cause a more severe form of DC than mutations in telomerase components. iv Acknowledgements Pursuing graduate school was not something I had anticipated till the third year of my undergraduate studies, so I feel a deep sense of gratitude towards a number of provocative mentors and supportive friends for invigorating this zeal for scientific research. I thank my thesis advisor, Roy, for leading by example on this pursuit of fundamental knowledge of our cells and their various minutiae. I have learned skills from Roy that will be useful not only in the lab, but also outside of it- asking the right questions, questioning conventional knowledge when necessary, and pushing yourself to be as efficient as you can be, to name a few. I also thank Roy for giving me a chance to spend a summer in his lab during my undergraduate studies, an opportunity that ultimately led to five years of graduate school culminating in this thesis. I thank my Master’s thesis advisor, Dr. P.I. Pradeepkumar, for mentoring me during my undergraduate studies at IIT Bombay, and providing me with a sound exposure to biological research, which was otherwise impossible to get in a core chemistry department. I also thank the former members of his lab, Dr. Kiran Gore, Dr. V. Dhamodharan and Dr. Jagdeesh Chandrashekhar, for teaching me the skills that were of great utility in graduate school. For an international student 9,000 miles away from the comfort of the native culture, the lab becomes family. I am extremely grateful to have had the good fortune of being a part of a supportive lab with talented and caring people all around me. It is a testament to the people here that we function more as a unit and less as a group of individuals pursuing their own research. I will cherish all the random chats about America and food, as well as the discussions and feedback over my research. I v especially want to thank two former members, Dr. Ross Buchan and Dr. Saumya Jain, for life-long memories and friendship. I thank the members of my thesis committee for being so supportive and helpful over the last two years, and providing me your valuable insight which has fostered new ideas and directions in my research. I especially want to thank Tom for his input and guidance during my study of human telomerase RNA quality control, which has now become a fruitful collaboration. I also thank the members of the Cech lab for their help during the course of this collaboration. Last but not the least, I want to express my love and gratitude to friends and family back home. Thank you mummy and papa for supporting me in all of my pursuits, even those that did not pan out. Thank you for never letting anything get in the way of my studies and research, even when times were hard. I thank my younger brother, Rishabh, for being the best sibling one could ask for. I am very proud of the person and the doctor he has become, and his hard work and dedication motivates me when things are not going smoothly. Finally, I thank my girlfriend Navneeta, for five wonderful years of companionship and love, which have been a source of comfort and inspiration. Life would not be what it is without you in it! vi Table of Contents Chapter 1: Hypo-assembly diseases of RNA-protein complexes .............................. 1 RNA quality control pathways compete with RNP assembly ................................. 1 Introduction ............................................................................................................... 1 Diversity of RNA quality control pathways in eukaryotes .......................................... 4 Hypo-assembly diseases of RNPs .......................................................................... 11 Dyskeratosis Congenita and degradation of telomerase RNA ................................ 11 Spinal Muscular Atrophy and snRNA degradation .................................................. 14 RNP hypo-assembly diseases include pathologies of ncRNAs ........................... 18 Hypo-assembly diseases of mRNPs ....................................................................... 20 Discovery of possible treatments for RNP hypo-assembly diseases ................. 23 Conclusion ................................................................................................................ 24 Chapter 2: Quality control of snRNAs in Spinal Muscular Atrophy ........................ 25 Summary ................................................................................................................... 25 SMA is a neurodegenerative disease caused by snRNP deficiency ................... 26 Results ....................................................................................................................... 28 Mutations in the yeast U1 snRNA Sm site destabilize the snRNA .......................... 28 Defective U1 snRNAs are degraded by both 3' to 5' exonuclease Rrp6 and decapping and Xrn1-mediated decay ..................................................................... 30 Decapping of defective U1 snRNAs in yeast is catalyzed by the Dcp2 enzyme ..... 36 Decapping and 5' to 3' degradation of defective snRNAs is conserved in mammalian cells...................................................................................................... 38 vii Reduction of snRNA levels by SMN knockdown can be rescued by inhibition of decapping and XRN1-mediated decay ................................................................... 42 Knockdown of DCP2 partially suppresses some SMN knockdown dependent splicing defects ........................................................................................................ 45 Discussion ................................................................................................................. 49 Assembly-defective snRNAs are subject to quality control ..................................... 49 Defects in snRNP assembly in mammals leads to decapping and 5' to 3' decay ... 49 Prevention of snRNA decapping may lead to rescue of snRNP function ................ 51 RNP biogenesis generally competes with quality control ........................................ 52 Experimental methods ............................................................................................. 53 Construction of U1 Sm-mutants in yeast and mammals ......................................... 53 Yeast strains and plasmids ....................................................................................
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