ARGLU1 is an RNA-Binding Protein that has a Fundamental Role in Modulating Global Alternative Splicing Patterns by Emma Zilberman A thesis submitted in conformity with the requirements for the degree of Master of Science Pharmaceutical Sciences University of Toronto © Copyright by Emma Zilberman 2017 ARGLU1 is an RNA-Binding Protein that has a Fundamental Role in Modulating Global Alternative Splicing Patterns Emma Zilberman Master of Science Pharmaceutical Sciences University of Toronto 2017 Abstract Alternative splicing (AS) is a key step of RNA maturation. More emerging evidence demonstrates RNA synthesis and processing are coupled, thus transcriptional coregulatory proteins are being studied in greater detail as mediators of both events. Nuclear receptors such as glucocorticoid receptor (GR) are ligand-dependent transcription factors dependent on coregualtor interaction. The role of GR in metabolism and immune function in response to synthetic glucocorticoids, such as Dexamethasone are well characterized. We identified a novel GR coactivator, arginine and glutamate rich 1 (ARGLU1) that potentiates GR transcriptional activity and interacts with splicing factors. ARGLU1 knockdown in a neuronal cell line and analysis of mRNA found ARGLU1 alters alternatively spliced events (ASEs). Dexamethasone-regulated ASEs were also identified and found to be largely dependent on ARGLU1 and GR. RNA immunoprecipitation found that ARGLU1 could bind pre-mRNA of ASEs. This thesis studied the role of ARGLU1 as a novel RNA-binding protein that modulates AS. ii Acknowledgments First of all, I am extremely grateful to my supervisor, Dr. Carolyn Cummins, for providing me the opportunity to work on a project so novel and unique that we both felt intrigued and sometimes so puzzled by the results. Beginning my thesis research was no easy task, and Carolyn’s passion for research helped ignite my own excitement for this work on splicing that I really grew to appreciate. Secondly, my advisory committee comprising of Dr. Stephane Angers and Dr. Craig Smibert, have been the perfect blend of critical, creative and supportive. Thank you for withstanding my often lengthy committee meetings and providing the help I needed to complete this work. To the Cummins lab, both past and present, you have made my time here fantastic! From the endless scientific advice to the hilarious, often very odd, discussions we all have. Lilia “my lab mom” – I cannot thank you enough for all the help you provided me throughout my MSc research, from teaching and explaining concepts to me repeatedly sometimes. Adil “my hilarious lab twin” – thank you for being so supportive both in the lab and out and helping teach me a lot of experimental techniques in the start. Paola, Cigdem, Michael and Wendy, you’re always around when I need a chat or coffee break. To past lab members, Ricky, Rucha and Jasmine – you were all great help whenever I needed advice. I would not be where I am without the help of my lab mates! To mom and dad, Daniel, and grandparents – you guys have relentlessly asked me what I do here and I never have a good answer, so I hope you enjoy this thesis to finally get an answer to your question! Your love, support and friendship has been unwavering. Thank you for the constant words of encouragement and praise. I love you all so much from the bottom of my heart! Dave, my love, my rock. You know me best of all and your words of encouragement through all the frustrating experimental setbacks and late night work was what really got me to make it to the end. I don’t think I would have ever been able to grow as a person and scientist without your unwavering motivation, love and support. iii Table of Contents Acknowledgments ........................................................................................................................ iii Table of Contents ......................................................................................................................... iv List of Tables ............................................................................................................................... vii List of Figures ............................................................................................................................. viii List of Appendices ........................................................................................................................ xi List of Abbreviations .................................................................................................................. xii Chapter 1 ........................................................................................................................................1 Introduction ...............................................................................................................................1 1.1 Nuclear receptors ........................................................................................................1 1.1.1 Glucocorticoid receptor ..................................................................................3 1.1.2 NR coregulators ..............................................................................................6 1.2 Constitutive and alternative splicing ..........................................................................6 1.2.1 Types of alternative splicing ...........................................................................7 1.2.2 Spliceosome components and mechanism ......................................................8 1.2.3 RNA sequences as a determinant of splicing ................................................12 1.2.4 Splicing factors as determinants of alternative splicing ...............................13 1.2.5 Positional context as a determinant of AS regulation ...................................15 1.2.6 Alternative splicing and nonsense-mediated decay (AS-NMD) ...................16 1.2.7 Alternative splicing in the nervous system ...................................................17 1.3 ARGLU1 ..................................................................................................................18 1.3.1 Discovery of ARGLU1 .................................................................................18 1.3.2 ARGLU1 C-terminal domain is a GR coactivator ........................................21 1.3.3 ARGLU1 N-terminal domain interacts with splicing factors .......................21 1.4 Co-transcriptional RNA processing ..........................................................................22 iv 1.4.1 Kinetic model of coupling ............................................................................24 1.4.2 Recruitment model of coupling ....................................................................24 1.4.3 NR coregulators: mediators of transcription and splicing coupling .............25 1.5 Objective of thesis research ......................................................................................26 1.5.1 Specific aims .................................................................................................26 1.5.2 Hypothesis ....................................................................................................27 Chapter 2 ......................................................................................................................................28 Methods ....................................................................................................................................28 2.1 Cell culture ...............................................................................................................28 2.2 Transfection assays ...................................................................................................28 2.3 RNA analysis, cDNA synthesis and real-time quantitative PCR (qPCR) ................28 2.4 Protein extraction ......................................................................................................29 2.5 BCA and WB ............................................................................................................29 2.6 RNA-seq analysis and validation .............................................................................29 2.7 Pathway analysis .......................................................................................................33 2.8 One-Step RT-PCR ....................................................................................................33 2.9 RNA binding motif analysis .....................................................................................35 2.10 RNA Immunoprecipitation (RIP) .............................................................................35 2.11 One-Step qPCR .........................................................................................................36 2.12 Statistical analysis .....................................................................................................37 Chapter 3 ......................................................................................................................................38 Results ......................................................................................................................................38 3.1 ARGLU1 modulates global patterns of gene expression .........................................38 3.1.1 RNA-seq gene expression validation ............................................................41 3.2 ARGLU1 modulates global patterns of AS ..............................................................45