WATCHING DYNAMICS AND ASSEMBLY OF SPLICEOSOMAL COMPLEXES AT SINGLE MOLECULE RESOLUTION A thesis submitted for the degree of DOCTOR OF PHILOSOPHY 2015 CHANDANI MANOJA WARNASOORIYA Section of Virology, Department of Medicine Imperial College London 1 Copyright Declaration The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. 2 Declaration of Origin I hereby declare that this project was entirely my own work (except the gel images in figures 4.2-4.5 in the chapter 4) and that any additional sources of information have been properly cited. I hereby declare that any internet sources, published and unpublished works from which I have quoted or drawn reference have been referenced fully in the text and in the reference list. I understand that failure to do so will result in failure of this project due to plagiarism. Chapter 3 was in collaboration with Prof. Samuel Butcher and Prof. David Brow at University of Wisconsin-Madison, Wisconsin, USA. The Prp24 full length protein and the truncated protein (234C) were provided by them. Chapter 4 was in collaboration with Prof. Kiyoshi Nagai and his lab members Dr. John Hardin and Yasushi Kondo at LMB Cambridge University, Cambridge, UK. All the Electrophoretic mobility shift assays for the full length U4/U6 complex and the calculations of binding affinities were done by Dr. John Hardin and Yasushi Kondo at LMB Cambridge University (Figures 4.2-4.5 in the chapter 4) and these figures were obtained from Dr. John Hardin and Yasushi Kondo with permission. 3 DEDICATION To My Loving Amma because you sacrificed all your wellbeing To give me a better life To my loving Thattha because you sacrificed all your happiness To make me happy To my loving husband because you sacrificed all your dreams To bring me all this long To my loving daughter because you sacrificed all your cuddling times To let me fulfil my task 4 ACKNOWLEDGEMENTS First of all I want to convey my sincere gratitude to my advisor, Dr David Rueda, for giving me the opportunity to work in his lab, which opens the path to learn an emerging technique and his guidance, encouragement and support throughout my research work. I’m fortunate to be a part of his lab and able to do promising research. Thank you very much Dr. Rueda for all the help and opportunities you have given me, and for the diligent effort you made to train me as a good scientist. Also I would like to thank him for giving me the opportunity to join and obtain my PhD from Imperial College London. I would also like to thank my thesis committee in Department of Chemistry, Wayne State University, USA, Prof. Louis Romano and Prof. Arthur Suits, for their valuable suggestions and feedback on my research during the first three years of my PhD. My special thanks go to Prof. Christine Chow for her cooperation, guidance and support through my difficult times. I express my gratitude to my research progress panel from section of Virology, Imperial College London, Prof. Wendy Barclay and Dr. Goedele Maertens for their valuable suggestions and scientific discussion during my early stage and late stage assessments. I would also like to thank Prof. Ramon Vilar and Dr. Ray O’Keefe for agreeing to be in my PhD viva committee. I am also thankful to our collaborators Prof. Samuel Butcher, Prof. David Brow and Ashley Richie from University of Wisconsin-Madison for sending me the Prp24 protein and important discussions and suggestions. Also Prof. Kiyoshi Nagai, Dr. John Hardin and Yasushi Kondo from LMB, Cambridge, UK for providing U4/U6 proteins, carried out the initial experiments and valuable discussions and suggestions. 5 Then, I would like to acknowledge all my lab members. My biggest thank goes to Zhuojun Guo for teaching me single molecule experiments and everything not only about research but also about life. Thank you so much Zhuojun for everything you taught me and for the fun times we had in the lab. I also want to give a big thanks to Dr. Amanda Solem for her continuous support and valuable suggestions and Dr. Elvin Aleman and Dr. Alfonso Brenlla for the technical support in single molecule setup. I also want to thank my former lab mates Rajan, Krishanthi, Rui, Sharla, May Eric, Pramodha, Gayan and Marcus for their immense guidance and support. I would also want to thank my fellow lab mates from Wayne State University, Bishnu, Hansini and Imali, for the valuable discussions we had and making the stressful graduate school life an endurable one. I will cherish all those lovely times that we spent together. I also like to thanks Chow lab members from Wayne State University for their help for my research, especially Gayani and Daya for helping me with MALDI and Xun for encouraging me and cheering me up in my difficult times. I would also like to thank my current lab members in Imperial College Sheila, Hailey, Kathy, Amit, Adam, Kotryna, Maria and Martin to all your help with my research work as well as with my personal life. Thank you very much Sheila, for being a wonderful friend to me and thank you for being there for me whenever I needed someone to talk in hard times. A special thank goes to Sheila, Hailey, Kathy and Adam for spending lot of time on reading and correcting my thesis, I really appreciate your help on that. Thank you all for making an enjoyable working environment. I want to convey my sincere appreciation to Wayne State University Chemistry department administrative staff specially Melissa Nestor, Debbie, Diane and all the 6 others. I would also like to thanks people in Imperial College who helped me in many ways. I would like to convey my sincere appreciation to everyone in the 10th floor, Commonwealth building; Guido lab members, Paul Coote, Cecilia George for all the help you gave. Also I would like to thank Hayley Kendall and Brett Onslow for helping me with the administrative work. Then my heartiest gratitude goes to the Wayne state - Sri Lankan student Association, Michigan Sri Lankan community and all my Sri Lankan friends, who help me to make a second home in USA. I’m thanking to all my best friends in Sri Lanka who were there for me to cheer me up and help me. Also I thank all my Sri-Lankan friends in London for making life in London much endurable one. Thank you being like my own family and helping me during my pregnancy and taking care of my baby during past year. Without you all it will not be easy for me to continue with my PhD while having baby. Finally, I would like to convey my heartfelt appreciation to my Parents; Upali Warnasooriya and Chandra Warnasooriya for bringing me up as a better human and for everything you did throughout my life. Also I thank to my brother Vindana and sister-in- law Pradeepa for encouraging me and also for taking care of our parents while I’m far away from them. Your love means a lot to me, especially to bear this stressful life in a foreign country. Then I want to express my utmost gratitude to guardian of my life, my loving husband Chinthaka Sanjeewa for your enormous support, encouragement and love which means so much for me. Since the day we met, you were there by my side for last 10 years, helping me in every way to fulfil my dreams and come this far in my career. Without your love, protection and guidance, I can’t imagine how I would have 7 come this far. Thank you very much for being with me in every step in my life and I’m sorry for all the hardship you have to face while you are helping me to accomplish my dreams. Last but not least I would like to thank my darling daughter, Chanudi who brought all the happiness to our lives. I’m really sorry that I couldn’t spend more time with you during your first year, the period you really need to be with your mother. Thank you for enduring the times that I’m away from you, working on the thesis all night without sleeping with you. I’m really blessed to have a lovely daughter as you, who never gave me a hard time and behave so nicely like you understand my situation. I love you so much my loving daughter. All these six years, I wouldn’t be able to focus on and successfully complete my PhD without all the help and support I had from all of you. Thank you all loving people !!! 8 TABLE OF CONTENT Copyright Declaration ................................................................................................... 2 Declaration of Origin ..................................................................................................... 3 DEDICATION .................................................................................................................. 4 ACKNOWLEDGEMENTS ............................................................................................... 5 TABLE OF CONTENT .................................................................................................... 9 TABLE OF FIGURES ................................................................................................... 16 LIST OF TABLES ......................................................................................................... 20 LIST OF ABBREVIATIONS .......................................................................................... 21 ABSTRACT .................................................................................................................