Characterization of the relationship between two RBM5 family members by Julie Jennifer Loiselle A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (PhD) in Biomolecular Sciences The Faculty of Graduate Studies Laurentian University Sudbury, Ontario, Canada © Julie Loiselle, 2017 THESIS DEFENCE COMMITTEE/COMITÉ DE SOUTENANCE DE THÈSE Laurentian Université/Université Laurentienne Faculty of Graduate Studies/Faculté des études supérieures Title of Thesis Titre de la thèse Characterization of the relationship between two RBM5 family members Name of Candidate Nom du candidat Loiselle, Julie Degree Diplôme Doctor of Philosophy Department/Program Date of Defence Département/Programme Biomolecular Sciences Date de la soutenance July 31,2017 APPROVED/APPROUVÉ Thesis Examiners/Examinateurs de thèse: Dr. Leslie Sutherland (Supervisor/Directeur(trice) de thèse) Dr. Eric Gauthier (Committee member/Membre du comité) Dr. Amadeo Parissenti (Committee member/Membre du comité) Approved for the Faculty of Graduate Studies Approuvé pour la Faculté des études supérieures Dr. David Lesbarrères Monsieur David Lesbarrères Dr. Doug Gray Dean, Faculty of Graduate Studies (External Examiner/Examinateur externe) Doyen, Faculté des études supérieures Dr. T.C. Tai (Internal Examiner/Examinateur interne) ACCESSIBILITY CLAUSE AND PERMISSION TO USE I, Julie Loiselle, hereby grant to Laurentian University and/or its agents the non-exclusive license to archive and make accessible my thesis, dissertation, or project report in whole or in part in all forms of media, now or for the duration of my copyright ownership. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also reserve the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that this copy is being made available in this form by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. ii Abstract RNA binding proteins (RBPs) control all aspects of RNA metabolism, and a single RBP can have numerous downstream effects. Alterations to their expression and/or function can, therefore, have remarkable consequences. For instance, decreased levels of the RNA binding motif domain (RBM) protein RBM5 are associated with increased risk of a number of cancer types, and RBM10 mutations can be lethal. Although these consequences are quite severe, little is known regarding the range of processes and events influenced by these two homologous RBPs. In fact, previous RBM5 and RBM10 functional studies were largely focused only on their abilities to promote two processes; apoptosis and cell cycle arrest. Potentially by control of these processes, RBM5 and RBM10 were shown to influence one event: differentiation. The objectives of this study were to identify all cellular processes and events enriched by changes in RBM5 and/or RBM10 expression in a particular cultured cell line, and to determine the extent of functional overlap for RBM5 and RBM10 in these cells. Towards these goals, a list of RBM5 and RBM10 mRNA targets and differentially expressed genes was determined using next generation sequencing techniques. Our data suggest that RBM5 and RBM10 do influence a wide range of cellular processes and events. Although there is overlap in RBM5 and RBM10 mRNA targets and differentially expressed genes, these RBPs can have antagonistic functions; for example our data suggest that RBM5 prevents the transformed state, whereas RBM10 actually promotes it in an RBM5-null environment. Furthermore, we present a working model by which RBM5 may regulate RBM10’s protransformatory function. Finally, we demonstrate a relationship between RBM5 and RBM10 in non-transformed cells. The results presented herein provide insight not only into the roles and regulation of RBM5 and RBM10, but of RBPs in general. Taken together, iii the results presented in the four papers included in this thesis expand the knowledge base of RBM5 and RBM10, which provides insight into the disease states associated with their disrupted expression or function. Our findings are thus relevant to a wide range of scientific fields including molecular, developmental and cancer biology. Keywords RNA binding proteins, RBM5, RBM10, RIP-Seq, RNA-Seq, Transcriptional studies, Small cell lung cancer, Myoblasts, Regulation of RNA binding proteins iv Co-Authorship Statement Chapter 2. Insight into the role of alternative splicing within the RBM10v1 exon 10 tandem donor site I determined RBM10 splice variant expression levels in GLC20 cells by RNA-Seq (Figure 2.3A). In addition, ST and I performed protein modeling. ST carried out all initial molecular biology experiments and is responsible for Figure 2.1D,E,F and Figure 2.2. CP performed the final RT- PCR experiment presented in Figure 2.1B. BK performed the final Western Blot presented in Figure 2.1Ci. JR performed the final NUMB RNA expression experiment, and produced the graph. AM performed the FISH analysis. LCS conceived the project and developed the model. ST prepared the first draft of the manuscript, and LCS wrote the final draft. Chapter 3. RBM5 reduces small cell lung cancer growth, increases cisplatin sensitivity and regulates key transformation-associated pathways I wrote the manuscript, and my results are presented in all 8 tables and Figures 3.3, 3.4, 3.5 (Sp1 section only), 3.6, 3.7, 3.8, 3.11, 3.12, 3.13, and 3.14. JR grew the GLC20 cells (parental and sublines) (Figure 3.1F) and performed cell growth and apoptosis assays (Figures 3.9 and 3.10). LCS established the stable RBM5-expressing GLC20 sublines and revised the manuscript. JL, JR and LCS conceived and designed the experiments. Additional acknowledged contributions are as follow; NRM characterized the GLC20 cell line (Figures 3.1 and 3.2), BK analyzed RBM5 antibodies (Figure 3.5), ST help prepare RNA-Seq samples and SH extracted RNA from the primary tissue specimens. Chapter 4. RBM10 promotes transformation-associated processes in small cell lung cancer and is directly regulated by RBM5 I wrote the manuscript, prepared all figures and was corresponding author. My results are included in both tables and Figures 4.1, 4.2, 4.3A,B, 4.4, 4.5, 4.6A,B,D, 4.7, 4.8. I also developed the model (Figures 4.9 and 4.10). JR established the stable RBM10KD clones, and performed Western blot and MTT assays (Figure 4.3C,D,E and 4.6C). LCS revised the manuscript. Chapter 5. Post-transcriptional regulation of Rbm5 expression in undifferentiated H9c2 myoblasts I wrote the manuscript, prepared all figures and was corresponding author. Some work was performed by me at the end of my masters, but specifically during my PhD studies I performed transient RBM5 knockdown experiments (two biological replicates) (Figure 5.2) and analyzed RBM5 overexpression experiments (Figure 5.3). I also contributed to the analysis of the stable RBM5KD clones (Figure 5.1) and designed the model (Figure 5.4). LCS established the stable RBM5KD clones and revised the manuscript. ST screened all stable RBM5KD H9c2 clones generated by LCS (Figure 5.1). v Acknowledgments I would first and foremost like to thank my supervisor, Dr. Leslie Sutherland. You have been an amazing mentor, and words cannot express my gratitude. Your dedication to the success of your students is unmatched, and has shaped me into the researcher I have become. Thank you for your friendship, patience, support and encouragement, which have helped me through the more difficult times and taught me how to approach and overcome all new challenges. Thank you to my committee members, Dr. Éric Gauthier and Dr. Amadeo Parissenti, for their insights, guidance and support throughout my doctoral studies. Thank you also to the past and present members of the Sutherland lab group, particularly, Sarah Hunt, Jose Knee and Justin Roy, for supporting me through the ups and downs of research, and making the lab such a friendly and happy environment. To all members of the Health Sciences North Research Institute, it’s been a pleasure working alongside such exceptional researchers. I am very appreciative of the funding I received from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Northern Cancer Foundation, which have allowed me to focus on research throughout my studies. A special thank you to my wonderful husband, Mathieu, who believes in me even when I don’t believe in myself. I will be forever grateful for your strong, unwavering love and support. Thank you also to Henri and Hugo, my two beautiful boys, who inspire me every day to be the best I can be. Finally, to my all of my family and friends, particularly my parents, I cannot thank you enough for your encouragement throughout all of my studies. Having such a strong support system has allowed me to dream big and accomplish this significant goal. vi Table of Contents Abstract .........................................................................................................................................