The Role of Arginine Methylation of Hnrnpul1 in the DNA Damage Response Pathway Gayathri Gurunathan
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The role of arginine methylation of hnRNPUL1 in the DNA damage response pathway Gayathri Gurunathan Faculty of Medicine Division of Experimental Medicine McGill University, Montreal, Quebec, Canada August 2014 A Thesis Submitted to McGill University in Partial Fulfillment of the Requirements for the Degree of Master of Science © Gayathri Gurunathan 2014 Abstract Post-translational modifications play a key role in mediating the DNA damage response (DDR). It is well-known that serine/threonine phosphorylation is a major post-translational modification required for the amplification of the DDR; however, less is known about the role of other modifications, such as arginine methylation. It is known that arginine methylation of the DDR protein, MRE11, by protein arginine methyltransferase 1 (PRMT1) is essential for the response, as the absence of methylation of MRE11 in mice leads to hypersensitivity to DNA damage agents. Herein, we identify hnRNPUL1 as a substrate of PRMT1 and the methylation of hnRNPUL1 is required for DNA damage signalling. I show that several RGG/RG sequences of hnRNPUL1 are methylated in vitro by PRMT1. Recombinant glutathione S-transferase (GST) proteins harboring hnRNPUL1 RGRGRG, RGGRGG and a single RGG were efficient in vitro substrates of PRMT1. Moreover, I performed mass spectrometry analysis of Flag-hnRNPUL1 and identified the same sites methylated in vivo. PRMT1-depletion using RNA interference led to the hypomethylation of hnRNPUL1, consistent with PRMT1 being the only enzyme in vivo to methylate these sequences. We replaced the arginines with lysine in hnRNPUL1 (Flag- hnRNPUL1RK) such that this mutant protein cannot be methylated by PRMT1. Indeed Flag- hnRNPUL1RK was undetected using specific dimethylarginine antibodies. Flag-hnRNPUL1RK did not co-immunoprecipitate with PRMT1, as expected, since PRMT1 is known to associate with its substrates. Flag-hnRNPUL1RK had reduced affinity to NBS1, a subunit of the MRE11- RAD50-NBS1, a DDR complex. Finally, Flag-hnRNPUL1RK had an aberrant localization at DNA damage breaks using laser microirradiation. Collectively, my findings provide insight into how the arginine methylation of hnRNPUL1 plays a significant role in the DNA damage response. 2 Sommaire Les modifications post-traductionnelles jouent un rôle fondamental, notamment dans la régulation des mécanismes de réponse aux dommages causés à l’ADN. Il a été démontré que la méthylation des arginines de la protéine MRE11 par l’enzyme protéine-arginine méthyltransférase 1 (PRMT1) est essentiel dans la voie de signalisation des dommages causés à l’ADN. Dans cette étude, nous décrivons l’identification d’un nouveau substrat de l’enzyme PRMT1 : la protéine hnRNPUL1. Dans un premier temps, nous avons identifié que la déplétion de l’enzyme PRMT1 induit une hypométhylation de la protéine hnRNPUL1. De plus la protéine hnRNPUL1 contient plusieurs motifs RGG/RG, une séquence consensus méthylée par les PRMTs. Un essai de méthylation in vitro a montré que plusieurs de ces séquences consensus sont méthylées par l’enzyme PRMT1. Une analyse in vivo de la protéine Flag-hnRNPUL1 par spectrométrie de masse a confirmée les sites de méthylation des motif RGG/RG identifiés précédemment in vitro. Ces résultats furent supportés par la génération d’un mutant Flag-hnRNPUL1RK dépourvue d’arginine dans les séquences RGG/RG méthylées identifiées. En utilisant un anticorps spécifique reconnaissant les arginines diméthylées, nous avons découvert que le mutant Flag- hnRNPUL1RK était hypométhylé. Nous avons également observé que le mutant ne pouvait plus interagir avec PRMT1 par co-immunoprécipitation, contrairement à la protéine non-muté Flag- hnRNPUL1. Ensemble ces résultats démontrent une association physique de la protéine hnRNPUL1 avec PRMT1 requise pour sa méthylation directe par PRMT1 sur les résidus arginines de ces motifs RGG/RG. 3 Nous avons ensuite découvert que le mutant hypométhylé Flag-hnRNPUL1RK présente une affinité de liaison réduite avec la sous-unité NBS1 du complexe de recrutement MRE11- RAD50-NBS1(MRN), impliqué dans la réponse aux dommages à l’ADN. En effet, nous avons été capable de co-immunoprécipiter la sous-unité NBS1 avec la protéine Flag-hnRNPUL1, mais pas avec son mutant Flag-hnRNPUL1RK. Finalement, la localisation de Flag-hnRNPUL1RK est aberrante en présence aux coupures des brins d’ADN. Nous pensons que ce mécanisme est régulé par la méthylation des motifs RGG/RG de hnRNPUL1 par PRMT1. De façon globale, ces résultats illustrent la façon dont la méthylation des arginines de la protéine hnRNPUL1 par l’enzyme PRMT1 joue un rôle de premier plan au niveau de la réponse aux dommages de l’ADN produite par les coupures doubles brins. 4 Preface This Master of Science thesis was written in accordance with the Guidelines for Thesis Preparation from the Faculty of Graduate Studies and Research of McGill University. The experiments and studies were conducted under the supervision of Dr.Stéphane Richard. As well, each contributor who has collaborated in this research is acknowledged in the following section and throughout the remainder of the text. The mass spectrometry analysis discussed in this thesis was performed by Dr. Éric Bonneil (Figure 6B). The laser scissor damage experiment discussed in this thesis was performed by Yan Coulombe in Dr. Jean-Yves Masson laboratory (Université de Laval) (Figure 9A). 5 Acknowledgements I would first like to extend my sincere gratitude and appreciation to my supervisor, Dr. Stéphane Richard, for giving me the remarkable opportunity to work on such an interesting project and guiding me every step of the way. His continuous support and encouragement has truly been the propelling force that has motivated and inspired me to learn various technical skills and explore the field without limiting myself, thereby allowing me to grow as a young scientist and pursue my studies at McGill University in an active and dynamic environment. I would also like to thank the members of my thesis advisory committee, who have provided me with valuable guidance and support while monitoring my progress throughout my studies. Their input and feedback throughout my annual committee meetings have been truly noteworthy and have enabled me to push myself a step higher in pursuing my scientific goals while constantly driving myself forward. My fellow colleagues (both past and present) in the Richard lab have provided me with immense support, guidance and care, which I am truly grateful for. Whether it is moral support during times of need, experimental expertise when troubleshooting experiments, or simply providing a friendly and exciting atmosphere to work in on a daily basis, I truly appreciate their support. I would like to extend my special thanks and sincere gratitude to Dr. Zhenbao Yu, who has provided me with continuous support in helping me develop the necessary skills required to perform various experiments and for his valuable input in the troubleshooting aspect of my experimental techniques. His guidance and experimental expertise has made a significant contribution to my project and allowed for the completion of my thesis. As well, my utmost gratitude goes out to Palaniraja Thandapani who has been extremely supportive in the 6 stimulation of scientific discussions and in providing moral support. All of my dear lab members have been the constant driving force in improving my research strategies in all aspects and their immense care, generosity and unconditional support has truly allowed me to acquire various skills and values both inside and outside the professional environment which are tools that shall be useful throughout my life. I also like to extend my thanks to the all the students, technicians and post-doctoral fellows who I have met at the Lady Davis Institute throughout my studies, for their insight into my project, moral support and encouragement. I would like to extend my gratitude to Dr. Éric Bonneil for performing the mass spectrometry analysis which has significantly contributed to my project. As well, the laser scissor damage research provided by Yan Coulombe under the guidance of Dr. Jean-Yves Masson has been crucial for the completion of my thesis and I would like to sincerely thank them for their valuable contribution to my project. I would like to acknowledge the Canadian Institutes of Health Canada (CIHR) for providing financial support for the research presented in this thesis. Last but not least, I would like to thank my parents and brother for being there for me and supporting me throughout my graduate studies and schooling. I am forever grateful for the love and care that they have provided me with. Their continuous support and motivation have always encouraged me to chase my dreams in life. 7 Table of Contents ABSTRACT ............................................................................................................2 SOMMAIRE ...........................................................................................................3 PREFACE ...............................................................................................................5 ACKNOWLEDGEMENTS ..................................................................................6 TABLE OF CONTENTS .......................................................................................8 LIST OF FIGURES AND TABLES ...................................................................10 LIST OF ABBREVIATIONS .............................................................................. 11 INTRODUCTION................................................................................................15