UNIVERSIDADE DE LISBOA FACULDADE DE MEDICINA DE LISBOA Transcription dynamics prevents RNA-mediated genomic instability through SRPK2-dependent DDX23 phosphorylation Sree Rama Chaitanya Sridhara Orientadores: Prof. Doutor Sérgio Alexandre Fernandes de Almeida Prof.ª Doutora Maria do Carmo Salazar Velez Roque da Fonseca Tese especialmente elaborada para obtenção do grau de Doutor em Ciências Biomédicas Especialidade Biologia Celular e Molecular 2017 UNIVERSIDADE DE LISBOA FACULDADE DE MEDICINA DE LISBOA Transcription dynamics prevents RNA-mediated genomic instability through SRPK2-dependent DDX23 phosphorylation Sree Rama Chaitanya Sridhara Orientadores: Prof. Doutor Sérgio Alexandre Fernandes de Almeida Prof.ª Doutora Maria do Carmo Salazar Velez Roque da Fonseca Tese especialmente elaborada para obtenção do grau de Doutor em Ciências Biomédicas especialidade Biologia Celular e Molecular Júri: Presidente: Professor Doutor José Luís Bliebernicht Ducla Soares, Professor Catedrático em regime de tenure e Vice-presidente do Conselho Cientifico da Faculdade de Medicina da Universidade de Lisboa. Vogais: - Doutor Nicholas Proudfoot, Professor of Molecular Biology da University of Oxford, Reino Unido; - Doutor Lars Erwin Theodoor Jansen, Investigador Principal do Instituto Gulbenkian de Ciência; - Doutor Luís Filipe Ferreira Moita, Investigador Principal do Instituto Gulbenkian de Ciência; - Doutor Claus Maria Azzalin, Investigador, Group Leader, do do Instituto de Medicina Molecular, unidade de investigação associada à Faculdade de Medicina da Universidade de Lisboa. - Doutor João António Augusto Ferreira, Professor Associado da Faculdade de Medicina da Universidade de Lisboa; - Doutor Sérgio Alexandre Fernandes de Almeida, Professor Auxiliar da Faculdade de Medicina da Universidade de Lisboa; (Orientador). Marie Skłodowska-Curie actions and Fundação para a Ciência e a Tecnologia 2017 “ A impressão desta tese foi aprovada pelo Conselho Científico da Faculdade de Medicina de Lisboa em reunião de dia 2017.05.23” As opiniões expressas nesta publicação são da exclusiva responsabilidade do seu autor Preface The ending is nearer than you think, and it is already written. All that we have left to choose is the correct moment to begin. Alan Moore The present thesis entitled ‘Transcription Dynamics Prevent RNA-Mediated Genomic Instability through SRPK2-Dependent DDX23 Phosphorylation’ is divided into five major chapters with a short summary preceding it in both Portuguese and English. Each chapter is divided into smaller sections that explain an experiment or an idea. A general introduction to the topic of research has been dealt in Chapter 1 with a special emphasis on ‘R-loops and RNA processing factors’. The results obtained pertaining to this dissertation are presented as three individual chapters − Chapter 2, 3 and 4. The central idea of this project is to comprehend the molecular mechanisms employed by cells to sense, signal and resolve R-loops. Each of Chapter 2, 3 and 4 will describe the data related to SRPK2 – signal transducer, DDX23 – molecular effector and RNA Pol II – sensor for R-loops, respectively. Extended data table 1 − SRPK1 and SRPK2 ChIP-seq data and Extended data table 2 – DDX23 mutational analysis were attached in electronic format. A general scope of the work with implications in cancer has been discussed in Chapter 5. All the technical details and materials used related to this study has been elaborately mentioned at the end as Appendix A in order not to break the flow of the main text. Additionally, references, abbreviations and published articles were mentioned at the end from Appendices B-D. All the files pertaining to this thesis are attached in electronic format for further reference. This thesis is profusely filled with paraphrases and anecdotes that emphasize the meaning of a chapter in order to provide an organic reading. Sree Rama Chaitanya Acknowledgments Thanks to my scientific mentor Prof. Sérgio de Almeida. I can never teach you anything, you will have to teach yourself, but I can help perhaps in giving expression to that thought. Vivekananda To learn by force or harshness; but direct them to it by what amuses their minds, so that you may be better able to discover with accuracy the peculiar bent of the genius of each. Plato Thanks to my co-supervisor Prof. Dr. Maria Carmo-fonseca and her group members for their kind support. Thanks to my past and present lab mates for a collective and friendly environment. Hearty thanks to all my well wishers in iMM. Thanks to my thesis committee members Dr. João Ferreira and Dr. Luis Moita for their valuable inputs. Immense thanks to all the people who read my thesis and gave valuable suggestions. Special thanks to all my collaborators for sharing reagents and protocols. Work of any sort is a collective effort, whether it is bones and flesh of our own or from others. Anonymous As friendly as people could be from worlds as different as theirs and ours at that particular moment in our lives. ‘Che’ Guevara Thanks to funding agencies Marie Skłodowska-Curie actions and Fundação para a Ciência e a Tecnologia. Thanks to iMM for the support. When there is wealth, a little effort accomplishes the task. Chanakya Making money isn't hard in itself... What's hard is to earn it doing something worth devoting one's life to. Carlos Ruiz Zafón. Thanks to Lisboa and Santarém :D. As nações todas são mistérios. Cada uma é todo o mundo a sós. Fernando Pessoa Thanks to my family for their love and support. Love is an endless mystery, because there is no reasonable cause that could explain it. Tagore In your love my salvation lies. Alexi Murdoch This work could not have been done without the love and support from the best part of my life − Friends! Depth of friendship does not depend on length of acquaintance. Tagore There is self-interest behind every friendship. There is no friendship without self-interests. This is a bitter truth. Chanakya friendship is a relationship that has no formal shape, there are no rules or obligations or bonds as in marriage or the family, it is held together by neither law nor property nor blood, there is no glue in it but mutual liking. It is therefore rare. Wallace Stegner Acknowledgments Contents RESUMO AND SUMMARY 9 CHAPTER 1. INTRODUCTION 13 1.1 GENOME INSTABILITY IS A HALLMARK OF CANCER 15 1.2 TRANSCRIPTION AS AN ENDOGENOUS SOURCE OF GENOME INSTABILITY – A FOCUS ON R-LOOPS 18 1.2.1 Biological roles of R-loops 20 1.2.1.1 R-loops and transcription 20 1.2.1.1.1 R-loops and transcription activation 20 1.2.1.1.2 R-loops and transcription termination 21 1.2.1.2 Role of R-loops in chromatin dynamics and genome organization 24 1.2.1.3 R-loops as drivers of genome instability 25 1.3 R-LOOPS AND DISEASE 27 1.3.1 Role of R-loops in neurological disorders 27 1.3.2 Role of R-loops in cancer 29 1.4 CELLULAR MECHANISMS TO MAINTAIN R-LOOP HOMEOSTASIS 29 1.4.1 Mechanisms to prevent deleterious R-loops 31 1.4.2 Mechanisms to resolve deleterious R-loops 32 1.5 SRPK1 AND SRPK2 AS CENTRAL MODULATORS OF RNA PROCESSING FACTORS 34 1.5.1 Functions of SRPK1 and SRPK2 35 1.5.2 Regulation of SRPK1 and SRPK2 36 1.5.3 Role of SRPK1 and SRPK2 in human malignancies 37 1.5.3.1 SRPKs in cancer 37 1.5.3.2 SRPKs in neuronal pathologies 37 1.6 OPEN QUESTIONS 39 CHAPTER 2. THE ROLE OF SRPK2 IN RNA-MEDIATED GENOME INSTABILITY 41 2.1 SRPK2 IS NECESSARY TO PROTECT THE GENOME INTEGRITY 43 2.2 RNA POL II ACTS AS A MOLECULAR BRIDGE FOR THE ASSOCIATION OF SRPK1 AND SRPK2 TO CHROMATIN 45 2.3 SRPK2 PREVENTS RNA-MEDIATED GENOME INSTABILITY 50 CHAPTER 3. THE DEAD BOX HELICASE DDX23 SUPPRESSES RNA- MEDIATED GENOME INSTABILITY 53 3.1 SRSF1 DOESNOT RESCUE DNA DAMAGE IN CELLS LACKING SRPK2 55 3.2 DEAD BOX HELICASES 56 3.2.1 Structure of DEAD box helicases 57 3.2.2 Mechanism of action of DEAD box helicases 58 3.2.3 DDX23 59 3.3 A PHOSPHOMIMETIC DDX23 RESCUES THE GENOME INTEGRITY IN SRPK2-DEPLETED CELLS 61 3.4 LOSS OF DDX23 DRIVES R-LOOP-DEPENDENT GENOME INSTABILITY 65 Contents CHAPTER 4. PAUSED RNA POL II ACTS AS AN R-LOOP 'SENSOR-SIGNAL' 69 4.1 DDX23 ACCUMULATES AT R-LOOP-CONTAINING CHROMATIN LOCI 71 4.2 RNA POL II PAUSING NUCLEATES SRPK2 AND DDX23 AT R-LOOP-CONTAINING LOCI 73 CHAPTER 5. DISCUSSION 77 5.1 SRPK2 AND DDX23 – NEW PLAYERS THAT PREVENT RNA-MEDIATED GENOME INSTABILITY 79 5.2 POTENTIAL LINK BETWEEN DDX23 AND ADENOID CYSTIC CARCINOMA 81 5.3 ROLE OF R-LOOPS IN DEFINING GENE BOUNDARIES 81 5.4 STALLED RNA POL II − A SENSOR FOR R-LOOPS 82 5.5 CONCLUDING REMARKS 84 APPENDIX A. MATERIALS AND METHODS 85 APPENDIX B. REFERENCES 97 APPENDIX C. ABBREVIATIONS 115 APPENDIX D. ARTICLES 119 Resumo and Summary Resumo Durante a transcrição, os transcritos de RNA são sintetizados pela RNA polimerase II (RNA Pol II) a partir da informação contida na cadeia de DNA. A síntese de RNA ocorre dentro da bolha de transcrição onde as duas cadeias de DNA são fisicamente separadas e o RNA nascente forma um híbrido RNA-DNA de aproximadamente 8 pares de bases com a cadeia molde. No entanto, a estrutura molecular da RNA Pol II assegura que o transcrito nascente seja fisicamente separado do DNA aquando da sua saída do local activo da enzima. Por esta razão, foi, até há pouco tempo, amplamente aceite que as estruturas híbridas de RNA-DNA seriam apenas produtos transientes da transcrição. Contudo, uma década de investigação demonstrou que híbridos de RNA-DNA se formam igualmente não é jusante da bolha de transcrição, originando, juntamente com cadeia de DNA desemparelhada, estruturas que se designam R-loops. Os R-loops são importantes para a regulação da dinâmica da transcrição e em diversos processos celulares, tais como a recombinação e a reparação de DNA. No entanto, os R-loops representam também uma grande ameaça à estabilidade genómica e devem por isso ser mantidos dentro de níveis fisiológicos. Em condições normais, as células regulam os níveis fisiológicos de R-loops através de enzimas como helicases de DNA/RNA e nucleases (como RNase H) de forma a separar ou digerir a fração de RNA dos R-loops, respetivamente, com o intuito de manter a conformação do DNA nativo. Além disso, são conhecidos vários fatores de processamento de RNA que impedem a formação de R-loops, sequestrando o RNA nascente e impedindo- o de se ligar ao DNA, prevenindo assim a instabilidade genómica associada à formação de R-loops. Por exemplo, o fator de ligação SRSF1 é conhecido por prevenir a instabilidade genómica mediada por R-loops. Contudo, as cascatas de sinalização e as vias moleculares que conduzem à sua mobilização e ativação são essencialmente desconhecidas. A atividade de diversos fatores de processamento de RNA é regulada através da fosforilação que é, em muitos casos, conduzida por proteínas quinase de serina/arginina (SRPK) 1 e 2. Estas quinases fosforilam domínios de arginina-serina (SR) de vários fatores de processamento de RNA classificados como proteínas-SR e regulam a sua função. O ciclo de fosforilação e desfosforilação prejudica a regulação da função das proteínas-SR e a sua ligação atempada aos elementos reguladores cis do RNA nascente. No entanto, a função das SRPK1 e SRPK2 no metabolismo de R-loops e na estabilidade do genoma ainda não foi desvendada. 9