ANALYSIS OF RBM5 AND RBM10 EXPRESSION THROUGHOUT H9C2 SKELETAL AND CARDIAC MUSCLE CELL DIFFERENTIATION by Julie Jennifer Loiselle A thesis submitted in partial fulfillment of the requirements for the degree of Master of Sciences (MSc) in Biology School of Graduate Studies Laurentian University Sudbury, Ontario © Julie Loiselle, 2013 THESIS DEFENCE COMMITTEE/COMITÉ DE SOUTENANCE DE THÈSE Laurentian Université/Université Laurentienne School of Graduate Studies/École des études supérieures Title of Thesis Titre de la thèse ANALYSIS OF RBM5 AND RBM10 EXPRESSION THROUGHOUT H9C2 SKELETAL AND CARDIAC MUSCLE CELL DIFFERENTIATION Name of Candidate Nom du candidat Loiselle, Julie Jennifer Degree Diplôme Master of Science Department/Program Date of Defence Département/Programme Biology Date de la soutenance July 15, 2013 APPROVED/APPROUVÉ Thesis Examiners/Examinateurs de thèse: Dr. Leslie Sutherland (Supervisor/Directrice de thèse) Dr. Céline Boudreau-Larivière (Committee member/Membre du comité) Dr. Éric Gauthier (Committee member/Membre du comité) Approved for the School of Graduate Studies Dr. Mazen Saleh Approuvé pour l’École des études supérieures (Committee member/Membre du comité) Dr. David Lesbarrères M. David Lesbarrères Dr. David A. Hood Director, School of Graduate Studies (External Examiner/Examinateur externe) Directeur, École des études supérieures ACCESSIBILITY CLAUSE AND PERMISSION TO USE I, Julie Jennifer 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 Motif (RBM) domain proteins RBM5 and RBM10 have been shown to influence apoptosis, cell cycle arrest and splicing in transformed cells. In this study, RBM5 and RBM10 were examined in non-transformed cells in order to gain a wider range of knowledge regarding their function. Expression of Rbm5 and Rbm10, as well as select splice variants, was examined at the mRNA and protein level throughout H9c2 skeletal and cardiac myoblast differentiation. Results suggest that Rbm5 and Rbm10 may (a) be involved in regulating cell cycle arrest and apoptosis during skeletal myoblast differentiation and (b) undergo post-transcriptional or translational regulation throughout myoblast differentiation. All in all, the expression profiles obtained in the course of this study will help to suggest a role for Rbm5 and Rbm10 in differentiation, as well as possible differentiation-specific target genes with which they may interact. Keywords RNA Binding Motif domain proteins; RBM5; RBM10; H9c2; Myoblast Differentiation; Alternative Splicing iii Acknowledgments First and foremost, I would like to thank my Supervisor, Dr. Leslie Sutherland. Throughout my masters you have given me the opportunity to learn and to experience the ins and outs of research, from proposal writing to paper submission, and for this I will be forever grateful. The passion and dedication you have towards your students’ learning and your research program are unmatched; I am very lucky to have been able to study and to grow under your guidance. Thank you also for your friendship and mentoring, which have helped me to successfully complete this research project and grow as a researcher. I would also like to thank my thesis committee members, Dr. Éric Gauthier, Dr. Céline Boudreau-Larivière and Dr. Mazen Saleh, for all of their time and valuable input regarding my master’s research project. I am fortunate to have had such a dedicated committee, and your perspectives on my work have undoubtedly made my project stronger. I am very lucky and grateful for the funding I received from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Ontario government, which has allowed me to dedicate myself entirely to this project for the past two years. Thank you to my lab mates Nina Rintala-Maki and Twinkle Masilamani for teaching me various molecular biology lab techniques and for helping me analyze and interpret my results. Thank you also to Sarah Tessier, who helped me optimize various experimental conditions. I am grateful to have been able to share the joys of successful experiments with all of you, and to have had your support and encouragement when those times were few and far between. In addition, thank you to all of the members of the iv Advanced Medical Research Institute of Canada, particularly the other past and present members of the Sutherland lab research group, who have helped me laugh through the more difficult days and made the lab feel like a second home. A special thank you to my wonderful husband, Mathieu Loiselle, for his unwavering support and faith in my abilities, I would not have been able to undertake this task without you by my side. Last but not least, thank you to my amazing family and friends who have encouraged me every step of the way and helped to keep me balanced and focused. The love and support I have received from of all of you means the world to me and has given me the strength to take on any new challenges. v List of Abbreviations ATRA All-Trans-Retinoic Ccid CASP-2 Caspase 2 CDK Cyclin Dependant Kinase CDKI Cyclin Dependant Kinase Inhibitor D0 to D7 Day zero to day seven of differentiation DM Differentiation Medium GM Growth Medium KD Knockdown LUST LUCA-15-Specific Transcript OE Overexpression PCR Polymerase Chain Reaction qPCR Quantitative Polymerase Chain Reaction Rb Retinoblastoma RBM RNA Binding Motif RT-PCR Reverse Transcriptase-Polymerase Chain Reaction TAE Tri-Acetate-EDTA TNF Tumour Necrosis Factor vi Table of contents Thesis Defence Committee ................................................................................................. ii Abstract .............................................................................................................................. iii Acknowledgments ............................................................................................................... iv List of Abbreviations .......................................................................................................... vi Table of contents ............................................................................................................... vii List of Figures ...................................................................................................................... x List of Tables ...................................................................................................................... xi Appendix Index ................................................................................................................. xii Chapter 1. Introduction ........................................................................................................ 1 1.1. RBM5 ............................................................................................................................... 2 1.1.1. Splice variants ....................................................................................................................... 2 1.1.2. Structure ................................................................................................................................ 5 1.1.3. Expression ............................................................................................................................. 5 1.1.4. Function ................................................................................................................................ 7 1.2. RBM10 ........................................................................................................................... 11 1.2.1. Splice variants ..................................................................................................................... 11 1.2.2. Structure .............................................................................................................................. 12 1.2.3. Expression ........................................................................................................................... 13 1.2.4. Function .............................................................................................................................. 13 1.3. Muscle differentiation model ......................................................................................... 15 1.3.1. Cell cycle arrest in muscle differentiation ........................................................................... 16 1.3.2. Apoptosis