Molecular basis for the recruitment and function of DDX6 and GIGYF2 for post-transcriptional silencing by miRNAs By Christopher Rouya Department of Biochemistry McGill University Montreal, Quebec, Canada August 2015 A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Masters of Science. ©Christopher Rouya, 2015 Dedication This thesis is dedicated to my mother. I love you and thank you. You have made me who I am, and will always be what inspires me to move forward. 2 Abstract Dynamic and localized changes in the proteome are often engendered by post-transcriptional pathways which rapidly alter the rates of mRNA translation and degradation. Specifically, the microRNA (miRNA) class of non-coding RNAs silence target mRNA transcripts through sequential mRNA translation suppression and decay. Despite a deeper understanding of the manner by which miRNAs commit their targets to the 5‟-3‟ mRNA degradation pathway, the molecular basis for translation inhibition by miRNAs remains unclear. Here, we have endeavoured to identify microRNA-induced silencing complex (miRISC)-associated proteins that may impact mRNA translation, and assess the functional relevance of these proteins for miRNA function. First, we determine that the translation repressor and decapping activator, DDX6, interacts with the mammalian miRISC via binding to the CCR4-NOT deadenylase complex component, CNOT1. Disrupting complex formation affects miRISC-dependent gene silencing, thus revealing how DDX6 is recruited to, and important for, optimal miRISC activity. Moreover, we discover that the miRISC core protein, GW182, directly binds a component of a putative translation repressor complex, GIGYF2. Abrogating this interaction is accomplished through targeted mutation of conserved elements in both proteins, and establishes the groundwork for future functional studies of this complex for miRNA repression. Hence, factors recruited to miRISC are identified, and their potential contributions to miRNA-directed control of gene expression are elucidated. 3 Resume Des changements dynamiques et localisés du protéome sont souvent engendrés par des mécanismes post-transcriptionnels qui modifient rapidement les taux de traduction et de dégradation des ARNm. En particulier, les petits ARN non-codants de type microARN (miARN) inhibent l‟expression de leur ARNm cibles en induisant de manière séquentielle la suppression de la traduction de ces ARNm, puis leur dégradation par la voie 5‟-3‟. Bien que le mécanisme de dégradation par les miARN soit bien compris, les bases moléculaires de l'inhibition traductionnelle de ces cibles restent encore inconnues. Nous avons donc cherché à identifier les protéines associées au complexe effecteur de l‟activité des miARN (miRISC) qui sont responsables de la répression traductionnelle, et à confirmer la pertinence fonctionnelle de ces protéines pour l‟activité des miARN. Nous avons ainsi déterminé que la protéine DDX6, un répresseur traductionnel et activateur du décoiffage des ARNm, interagit avec le complexe miRISC via sa liaison à la sous-unité CNOT1 du complexe de déadénylation CCR4-NOT. L‟invalidation de l‟interaction DDX6-CNOT1 perturbe la répression par le complexe miRISC, confirmant donc que DDX6 est indispensable à l'activité optimale de miRISC. De plus, nous avons découvert que la protéine centrale de miRISC, GW182, se lie directement à la protéine GIGYF2, un composant d'un complexe répresseur putatif de la traduction. Des expériences de mutagénèse dirigée visant les résidus conservés sont actuellement en cours afin d‟inhiber l‟interaction GW182-GIGYF2. Ces données permettront d‟établir des bases expérimentales solides pour de futures études fonctionnelles du rôle de ce complexe dans la répression par les miRNA. Par conséquent, de nouveaux facteurs recrutés par le complexe miRISC ont été identifiés, et leurs contributions potentielles à la régulation de l‟expression génique par les miARN sont désormais mieux comprises. 4 Preface This thesis is a compilation of one published manuscript for which I am first author, and ongoing work for a manuscript that is in preparation. Chapter 2: Human DDX6 Human DDX6 effects miRNA-mediated gene silencing via direct binding to CNOT1. Christopher Rouya, Nadeem Siddiqui, Masahiro Morita, Thomas F. Duchaine, Marc R. Fabian, and Nahum Sonenberg. RNA 2014 Sep; 20(9): 1398-409 Chapter 3: Investigating the functional significance of a GW182-GIGYF2 interaction for post- transcriptional gene silencing by miRNAs. Christopher Rouya, Nadeem Siddiqui, Masahiro Morita, Nahum Sonenberg (In preparation) I would like to acknowledge the work of my co-authors for each chapter, and thank them for their collaboration. Chapter 2 Nadeem Siddiqui provided invaluable guidance in developing the project and preparing the manuscript. Masahiro Morita aided in the interpretation of RT-qPCR data. Marc R. Fabian gave critical insights on technical and conceptual aspects of the paper, and helped to direct the project. Thomas F. Duchaine provided expert comments on the manuscript. I performed all experiments and wrote the entire manuscript. Chapter 3 Masahiro Morita helped conceive the project, and provided a great deal of guidance along with Nadeem Siddiqui. I performed all experiments for this chapter. 5 Acknowledgements I would like to extend my sincerest gratitude towards Dr. Sonenberg for allowing me to work in his laboratory and benefit from his training. In this short time, your expectations, constant questioning, and passion for research have enabled me to grow dramatically as a young scientist, and have motivated me to continue investigating fundamental biological questions. I have learned invaluable lessons about how to survive and thrive in an incredibly competitive environment from our interactions, and I appreciate everything you have done for me. Being surrounded by talented and bright researchers has contributed greatly to my graduate studies, and I am grateful to all members of the laboratory for their support at one time or another. I am especially indebted to several of my colleagues who have been instrumental in forwarding the projects that I have been a part of, and who have been there for encouragement and experimental help along the way. To Nadeem Siddiqui, I “thank you very much” for everything: the wisdom, the discussions, the science, the help, and the laughs. I am not sure what this all would have been like without someone around who recognizes strangeness the way I do, so I am extremely happy to have shared this experience with you. To Marc Fabian, you taught me everything I know about how to practically approach science and writing. You were an excellent mentor, and consistently acted as an example for what an effective scientist should be. You showed me that balancing experiments, writing, presentations, and life can be possible, so long as you (presumably) never sleep. I will always remember your training, and deeply appreciate your influence on my growth as a scientist. To Masahiro Morita, I could not ask for a better desk mate. I remain convinced that you have done every experiment known to man, and I hope to become as capable and as brilliant of a scientist as you are. I thank you immensely for your patience in listening to my experimental issues, and for providing great ideas for my projects. I must thank Meena Vipparti and Sandra Perreault for their excellent technical assistance. Without them, our lab would cease to function. I would also like to acknowledge people who gave me general guidance and made the laboratory an enjoyable place to spend 60 hours a week. In particular, Evette Yassa, Johannes Ristau, Maayan Shapiro, Yuri Svitkin, Chadi Zakaria, Nathaniel Robichaud, Clement Chapat, Akiko Yanagiya, Tommy Alain‟s singing voice, Devon Merkley, and Soroush Tahmasebi have all contributed to an amazing laboratory atmosphere. 6 Table of Contents Dedication ....................................................................................................................................... 2 Abstract ........................................................................................................................................... 3 Resume ............................................................................................................................................ 4 Preface............................................................................................................................................. 5 Acknowledgements ......................................................................................................................... 6 Table of Contents ............................................................................................................................ 7 List of Figures ................................................................................................................................. 9 List of Tables .................................................................................................................................. 9 1 Chapter 1: Introduction .......................................................................................................... 10 1.1 Post-transcriptional control ............................................................................................ 10 1.2 Overview of Eukaryotic mRNA translation ................................................................... 12 1.3 The Eukaryotic Ribosome .............................................................................................