Characterizing the Telomerase-associated and H/ACA Ribonucleoprotein Dyskerin: Regulation and Function Deanna MacNeil Department of Anatomy and Cell Biology, McGill University, Montreal April 2020 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy in Cell Biology © Deanna MacNeil 2020 Table of Contents Abstract (English) ........................................................................................................................... 4 Résumé (French) ............................................................................................................................. 6 Acknowledgements ......................................................................................................................... 8 Accepted and Submitted Manuscript Included in this Thesis ....................................................... 11 Other Contributions by Candidate ................................................................................................ 11 Author Contributions by Section .................................................................................................. 12 Contributions to Original Knowledge ........................................................................................... 12 Abbreviations ................................................................................................................................ 14 Figures List ................................................................................................................................... 20 Introduction ................................................................................................................................... 22 Chapter 1: Literature Review ........................................................................................................ 24 1.1 Telomeres and Telomerase ................................................................................................. 24 1.1.1 DNA Protection ........................................................................................................... 28 1.1.2 Telomerase Recruitment .............................................................................................. 28 1.1.3 Telomerase Activity and Stimulation .......................................................................... 30 1.2 hTR Biogenesis and Processing .......................................................................................... 32 1.2.1 hTR and the Spliceosome ............................................................................................ 37 1.2.2 hTR and the Exosome .................................................................................................. 37 1.2.3 A Processing Role for PARN ...................................................................................... 41 1.3 Dyskerin, H/ACA RNAs and the H/ACA Complex ........................................................... 43 1.3.1 The H/ACA RNPs........................................................................................................ 47 1.3.2 H/ACA RNP Assembly ............................................................................................... 48 1.3.3 The H/ACA RNP and Pseudouridine Synthesis .......................................................... 51 1.3.4 The H/ACA RNP and Telomerase ............................................................................... 53 1.4 Dyskerin Function and Localization ................................................................................... 62 1.4.1 Membrane-free Subnuclear Compartmentalization: Nucleoli and Cajal Bodies ......... 62 1.4.2 Posttranslational Modifications and LLPS .................................................................. 67 1.5 SUMOylation Orchestrating Ribosomes and Telomere Maintenance................................ 74 1.5.1 Ribosome Biogenesis and SUMOylation .................................................................... 75 1.5.2 Telomere Maintenance and SUMOylation .................................................................. 77 1.6 Telomerase RNP and Premature Aging Disease ................................................................ 79 1.6.1 H/ACA complex and assembly factors in premature aging disease ............................ 80 2 1.6.2 RNA surveillance machinery in premature aging disease ........................................... 81 Chapter 2 – N-terminal residues of human dyskerin are required for interactions with telomerase RNA that prevent RNA degradation ............................................................................................. 83 2.1 Preface............................................................................................................................. 83 2.2 Title Page ........................................................................................................................ 84 2.3 Abstract ........................................................................................................................... 85 2.4 Introduction ..................................................................................................................... 85 2.5 Materials and Methods .................................................................................................... 89 2.6 Results ............................................................................................................................. 93 2.7 Discussion ..................................................................................................................... 112 2.8 Supplemental Figures and Figure Legends ................................................................... 117 2.9 Acknowledgements ....................................................................................................... 123 2.10 Author Contribution .................................................................................................... 123 2.11 References ................................................................................................................... 123 Chapter 3 – SUMOylation- and GAR1-dependent regulation of dyskerin nuclear and subnuclear localization .................................................................................................................................. 129 3.1 Preface........................................................................................................................... 129 3.2 Title Page ...................................................................................................................... 130 3.3 Abstract ......................................................................................................................... 131 3.4 Introduction ................................................................................................................... 131 3.5 Materials and Methods .................................................................................................. 134 3.6 Results ........................................................................................................................... 139 3.7 Discussion ..................................................................................................................... 155 3.8 Supplemental Figures and Figure Legends ................................................................... 160 3.9 Acknowledgements ....................................................................................................... 173 3.10 Author Contribution .................................................................................................... 173 3.11 References ................................................................................................................... 173 Chapter 4: Discussion ................................................................................................................. 180 Reference List ............................................................................................................................. 188 3 Abstract (English) The aim of this thesis is to understand how the function of the H/ACA ribonucleoprotein and telomerase component dyskerin is impacted in premature aging disease, and how dyskerin is regulated at a fundamental level. The H/ACA structural motif is present in many different non- coding RNAs, which assemble with the H/ACA ribonucleoprotein (RNP) complex involving dyskerin, NOP10, NHP2, and GAR1. Assembly with the H/ACA RNP is important for biogenesis of H/ACA RNA, thus dyskerin and the other RNP components play important roles in diverse cellular functions, including regulation of the ribosome, spliceosome, and human telomerase. Dyskerin is a pseudouridine synthase which catalyzes the posttranscriptional modification of ribosomal RNA and small nuclear RNA, guided by H/ACA RNAs. As it pertains to telomerase, the human telomerase RNA (hTR) component bears an H/ACA RNA biogenesis motif, making dyskerin essential for mediating hTR levels and telomerase activity. The importance of dyskerin to telomerase is underlined by mutations in the gene encoding dyskerin, which cause the premature aging disease X-linked dyskeratosis congenita (X-DC). This disease is caused by the accelerated shortening of DNA ends (telomeres). Telomeres are normally maintained by telomerase in stem cells and germ cells, but instead undergo progressive shortening in X-DC patients due to reduced hTR levels, impacting