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Investigation of the physiological roles of SRSF1-mediated translation Fiona Haward Doctor of Philosophy The University of Edinburgh 2017 Declaration I declare that this thesis is a result of my own work, unless otherwise stated, where the contributor has been fully acknowledged. This thesis has not been submitted in any form for any degree, qualification or similar at any other University or Institution. Fiona Haward, September, 2017 Acknowledgements The past four years have been some of the most exciting and enjoyable, but also some of the most challenging. The work undertaken to complete this thesis would not have been even half possible without the wealth of support I have received from numerous people, both within the IGMM and closer to home. Firstly, I would like to thank my supervisor Javier Cáceres for persuading me to embark on this project, when, in his words, he could convince no other to do so. Throughout which he has provided invaluable scientific direction and discussion regarding a field to which his knowledge is unparalleled. Of course, I also have to mention the infamously well-catered, and sometimes long, Cáceres Lab meetings! I also owe a huge amount of gratitude to Magdalena Maslon, who has provided me with the most incredible support during my PhD and is instrumental in the huge amount I have learnt during this time; it has been a privilege to work alongside her. She was particularly patient and caring when the project was tough or I just didn’t understand, especially in certain (mathematical!) departments. I would also like to thank the other members of the Cáceres Lab, past and present, for all the help, enjoyment and answers to obscure questions that they have provided over the years. I would especially like to acknowledge Dasa Longman for the excellent lab and life advice, the latter has guided me in many a low moment. Elsewhere within the IGMM, I would like to acknowledge Ian Adams for collaborating with the mice work for this project and providing an incredible knowledge for mouse development and genetics. Furthermore, I would like to thank Dr Andrew Wood for all the CRISPR advice during the project, without which, progress would have fallen short. On a personal note, I would like to thank Ailsa Revuelta, Katy McLaughlin, Flora Dix and Louise Cleal, who have provided countless laughs, and Christine Mordstein for the early morning discussions, which (occasionally) featured RNA. Also to Adam Douglas, whose encouragement, kindness and support was unrivalled at times when I needed it the most. Finally, I would like to thank my family, in particular my parents and brother, whose unwavering care, support and humour (and tolerance of constant phonecalls!) has been more than I could have wished for, I owe much of this effort to them. i Abstract The serine/arginine-rich (SR-) family proteins constitute a diverse group of pre- mRNA splicing factors that are essential for viability. They can be characterised based on the presence of one or two RRMs and an RS domain. A subset, of which SRSF1 is the prototype, is capable of nucleocytoplasmic shuttling; a process governed by continual cyclic phosphorylation of the RS domain. In contrast, SRSF2, another member of the SR family, is unable to shuttle due to the presence of a nuclear retention sequence (NRS) at the C-terminus of its RS domain. When this NRS is fused to SRSF1, it prevents nucleocytoplasmic shuttling of the SRSF1-NRS fusion protein. In addition to its nuclear roles, SRSF1 is directly associated with the translation machinery and can activate mRNA translation of target transcripts via an mTOR-dependent mechanism. The specific mRNA translational targets that SRSF1 serves to regulate encode numerous factors including RNA processing factors and cell-cycle proteins. The aim of this work is to study the physiological relevance of SRSF1 cytoplasmic functions, as previous data have relied on overexpression systems. CRISPR/Cas9 editing was used to knock-in the NRS naturally present in SRSF2 at the SRSF1 genomic locus, creating an SRSF1-NRS fusion protein. After numerous attempts, it was only possible to obtain a single viable homozygous clone in mouse embryonic stem cells (mESCs), despite being able to successfully tag the genomic SRSF1 locus. This strongly suggests that the ablation of SRSF1 shuttling ability is highly selected against in mESCs. To assess the physiological importance of SRSF1 nucleocytoplasmic shuttling during development, a mouse model for SRSF1-NRS was also developed. SRSF1-NRS homozygous mice are born at correct Mendelian ratios, but are small in size and present with severe hydrocephalus. Finally, proteomics was used to identify interactors of endogenous cytoplasmic SRSF1 and those that bind the NRS of SRSF2 to gain insights into the mechanism of nuclear retention for non- shuttling SR proteins. In summary, this work analyses the physiological relevance of cytoplasmic SRSF1 function and the consequences of the SRSF1-NRS allele in mouse development. ii Lay Abstract Every cell of an organism contains the same genetic material consisting of DNA. DNA contains units known as genes, which are transcribed, processed, and eventually expressed as proteins that execute numerous cellular functions. Interspersed within the protein-coding DNA, termed exons, some sequence does not encode for protein. These sequences are called introns. During transcription, the full DNA sequence of the gene is copied into a molecule called messenger RNA (mRNA), which is the template for protein synthesis. Therefore, to produce a continuous, functional mRNA transcript, the cell removes the introns and joins the exons together in a process called mRNA splicing. To perform splicing, the cell requires certain proteins called splicing factors. One essential splicing factor is SRSF1, which can regulate specific mRNA transcripts. The distinct molecular machineries required to produce mRNA or protein molecules are in different compartments of the cell. First, mRNA is synthesised and spliced in the nucleus and is then exported into the cytoplasm to be translated into protein. SRSF1 is an unusual splicing factor with multiple roles in gene expression. In the nucleus, SRSF1 promotes mRNA splicing, and in the cytoplasm, it facilitates translation of this same spliced mRNA into protein. However, the role of SRSF1 in protein synthesis is not well understood. This work aims to investigate the roles of SRSF1 function in protein synthesis and its relevance for physiology. Both cellular and whole organism models have been engineered to create a version of SRSF1 that is permanently retained in the nucleus and therefore unable to move to the cytoplasm to act on protein synthesis. This model has been used to investigate the physiological consequences on gene expression when SRSF1 is retained in the nucleus. iii Table of Contents Declaration .................................................................................................... i Acknowledgements ...................................................................................... i Abstract ........................................................................................................ ii Lay Abstract ................................................................................................ iii List of Figures ........................................................................................... viii List of Tables ................................................................................................ x List of Abbreviations .................................................................................. xi Introduction .................................................................................................. 1 1.1 Gene expression regulation and RNA binding proteins ........................................ 2 1.2 Pre-mRNA Splicing .......................................................................................................... 3 1.2.1 The Spliceosome and the splicing cycle .............................................................. 4 1.2.1.1 Components of the Spliceosome ..................................................................... 5 1.2.1.2 Spliceosome Assembly and Mechanistic Outline ................................................ 6 1.3 Splicing Regulation.................................................................................................... 9 1.3.1 Alternative splicing ............................................................................................... 9 1.4 SR Proteins ............................................................................................................... 11 1.4.1 Structure