Mediators of pre-mRNA splicing regulate sister chromatid cohesion in mammalian cells Sriramkumar Sundaramoorthy University College London and Cancer Research UK London Research Institute PhD Supervisor: Dr. Mark Petronczki A thesis submitted for the degree of Doctor of Philosophy University College London January 2014 Declaration I Sriramkumar Sundaramoorthy confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract The ‘endless forms most beautiful’ that populate our planet rely on the process of cell division to ensure equal segregation of the cellular content including the DNA to the two daughter cells. The accurate segregation of chromosomes in eukaryotes relies on connection between replicated sister chromatids, a phenomenon known as sister chromatid cohesion. Sister chromatid cohesion is mediated by a conserved ring-like protein complex known as cohesin. Defects in this process can promote aneuploidy and contribute to meiotic segregation errors with adverse consequences for developing embryos. Despite numerous advances into understanding cell division at the molecular level, we still lack a comprehensive list of the participating proteins and complexes. The aim of this thesis was to use available functional genomic and proteomic data to identify novel regulators of mitosis in human cells. Using an RNAi approach, we identified a set of factors involved in pre-mRNA splicing whose depletion prevents successful cell division. Loss of these splicing factors leads to a failure in chromosome alignment and to a protracted mitotic arrest that is dependent on the spindle assembly checkpoint. This mitotic phenotype was accompanied by a dramatic loss of sister chromatid cohesion that we could show happens as soon as DNA replication. While depletion of pre-mRNA splicing mediators had no effect on cohesin loading onto chromatin, it prevented the stable association of cohesin with chromatin. Immunoblotting revealed that the depletion of splicing factors caused a 5-fold reduction in the protein levels of Sororin, a protein required for stable association of cohesin with chromatin in post-replicative cells. Further analysis suggests erroneous splicing of Sororin pre-mRNA upon depletion of splicing factors. Importantly, the sister chromatid cohesion loss caused by depletion of splicing factors could be suppressed by a Sororin transgene that lacks introns. Our results suggest that that pre-mRNA splicing of Sororin is a rate-limiting step in the maintenance of sister chromatid cohesion in human cells. Our work reveals that a primary cellular pathology of compromised pre-mRNA splicing is a mitotic arrest accompanied by split sister chromatids. Our work linking splicing and sister chromatid cohesion has implications for the pathology of Chronic Lymphocytic Leukemia (CLL). One of the splicing factors that we implicate in sister chromatid cohesion is SF3B1, whose gene is one of the most frequently mutated genetic drivers found in CLL patients. 3 Acknowledgement At the outset, I would like to thank my supervisor Mark Petronczki for providing me not just with an opportunity to work with him but also to share his knowledge and experience about working in cell biology. I am deeply grateful to Mark for having taught me the importance of both doing science with integrity and passion while equally importantly being able to present your ideas and work in as lucid a form as possible. I would also like to thank him for instilling in me the importance for any experiment either in the lab or in life in general to have controls. I am thankful to the people in the Cell Division and Aneuploidy (CDA) lab for all their help and guidance over the years. It was a privilege to be surrounded by people smarter than myself so that I could learn as much as I have. I would like to thank Lola for being a great sounding board for all my ideas about science, theology, politics etc. and also for all her help with the many experiments. I would like to thank Sergey ‘Tovarich’ Lekomtsev for introducing me to the dark room 4 years ago and less importantly for helping me with my screen and for all the help over the past 4 years. I am grateful to Kuan-Chung for his thoughts about experiments, science, life and his ‘inspiring views’ about life and the food in London. Thanks go out to Tohru for his great guidance and for having had to endure my umpteen questions when I started out in the lab. I would also like to thank Laurent for being a brilliant ‘lab elder brother’, pointing out when I was doing wrong to make me a better researcher but also being among the first to compliment me on my rare good days. Special thanks go to Kristyna for all her help in and out of the lab over the past couple of years and for sharing all the wonderful stories about the 398 buses. I would also like to thank Murielle for her constant encouragement, help with experiments and for all the rides to Potters Bar. I am thankful to Peter Parker and Mike Howell for spending time away from their busy schedule on my thesis committee providing their valuable guidance and suggestions at every stage of my PhD at the LRI. Special thanks go to Mike and the members of the High Throughput Screening lab for their help in performing my screen 4 I am also indebted to all the people in Clare hall for making it a wonderful place to work in. Special thanks go to Marco for his help with the RT-PCR experiments, Nicola and Katharina for their help and guidance when I was starting out at Clare Hall. I am also grateful to the research administrators and assistants both at Clare hall and Lincoln’s inn fields for their excellent help in terms of my umpteen requests for visas, letters and accommodation etc. Without their help, the PhD would have easily been a much more arduous experience. I would also like to thank my friends and family for their steadfast support not just through my PhD years but also throughout my life. I am thankful to the Breast Cancer Campaign for providing me with a PhD grant for the duration of my PhD and for the special opportunity to visit the British houses of Parliament. Finally, I would like to thank Prof. Richard Dawkins for having fostered in me a sense of curiosity and wonder about the natural world and for instilling in me the perseverance to question dogmas and blind faith. 5 Table of Contents Abstract ............................................................................................................... 3 Acknowledgement .............................................................................................. 4 Table of Contents ................................................................................................ 6 Table of figures ................................................................................................... 9 List of tables ...................................................................................................... 12 Abbreviations .................................................................................................... 13 Chapter 1. Introduction .................................................................................. 15 1.1 The cell cycle ....................................................................................................... 15 1.1.1 Cell cycle phases and checkpoints ................................................................ 15 1.1.2 Regulation of the cell cycle ........................................................................... 18 1.1.3 Mitosis in mammalian cells .......................................................................... 22 1.1.4 Multiple supervisory mechanisms regulate mitosis ...................................... 25 1.2 Sister chromatid cohesion .................................................................................. 32 1.2.1 Sister chromatid cohesion is mediated by a protein complex called cohesin 33 1.2.2 Cohesin structure ........................................................................................... 34 1.2.3 Models for cohesin mediated sister chromatid cohesion .............................. 38 1.2.4 Cohesin loading onto chromatin involves the Adherin complex, the opening of the SMC1-SMC3 hinge and cohesin’s ATPase activity ....................................... 40 1.2.5 Establishment of sister chromatid cohesion happens during DNA replication .................................................................................................................. 42 1.2.6 Sororin plays a very important role in maintenance of cohesion in post- replicative cells .......................................................................................................... 44 1.2.7 Cohesin removal from mitotic chromosome happens in two distinct steps in higher eukaryotes ...................................................................................................... 46 1.2.8 Roles of cohesin beyond sister chromatid cohesion ..................................... 52 1.2.9 Cohesinopathies and cohesin in cancer ......................................................... 56 1.3 Pre-mRNA splicing ............................................................................................ 58 1.3.1 The spliceosome and pre-mRNA splicing .................................................... 59 1.3.2 Models of pre-mRNA splicing and cis-acting pre-mRNA elements in splicing .....................................................................................................................