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Investigation of RNA Binding Proteins Regulated by Mtor Investigation of RNA binding proteins regulated by mTOR Thesis submitted to the University of Leicester for the degree of Doctor of Philosophy Katherine Morris BSc (University of Leicester) March 2017 1 Investigation of RNA binding proteins regulated by mTOR Katherine Morris, MRC Toxicology Unit, University of Leicester, Leicester, LE1 9HN The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase which plays a key role in the transduction of cellular energy signals, in order to coordinate and regulate a wide number of processes including cell growth and proliferation via control of protein synthesis and protein degradation. For a number of human diseases where mTOR signalling is dysregulated, including cancer, the clinical relevance of mTOR inhibitors is clear. However, understanding of the mechanisms by which mTOR controls gene expression is incomplete, with implications for adverse toxicological effects of mTOR inhibitors on clinical outcomes. mTOR has been shown to regulate 5’ TOP mRNA expression, though the exact mechanism remains unclear. It has been postulated that this may involve an intermediary factor such as an RNA binding protein, which acts downstream of mTOR signalling to bind and regulate translation or stability of specific messages. This thesis aimed to address this question through the use of whole cell RNA binding protein capture using oligo‐d(T) affinity isolation and subsequent proteomic analysis, and identify RNA binding proteins with differential binding activity following mTOR inhibition. Following validation of 4 identified mTOR‐dependent RNA binding proteins, characterisation of their specific functions with respect to growth and survival was conducted through depletion studies, identifying a promising candidate for further work; LARP1. Having selected LARP1 from depletion screens, overexpression co‐IP experiments conducted alongside known binding partner PABP and subsequent arrays allowed for preliminary identification of mRNAs to which LARP1 binds. Finally, we showed evidence for differential binding of mRNA subsets between LARP1 and PABP, opening a new caveat for the role of the effector protein LARP1 in mTOR dependent gene expression regulation. 2 Acknowledgements First and foremost, I would like say a massive thank you to Professor Martin Bushell for not only giving me the opportunity to do this PhD, but also for his incredible and unwavering support, encouragement and patience over the last four years in his lab. My eternal gratitude goes also to Dr Ewan Smith for being an amazing mentor, colleague and friend throughout the life of this project. Special thanks to Dr Ania Wilczynska for all of her guidance and encouragement; to Dr David Ferland‐ McCollough for teaching me many of the initial methods I learnt; and to Dr Jack Godfrey, for never failing to keep me grounded. A huge thank you to the members of the Bushell Lab past and present, for the advice, laughs and support, but most of all for making it a truly wonderful place to be, even when it seemed science was determined to ruin my day. Thank you to the proteomics group led by my secondary supervisor Professor Kelvin Cain; in particular to Mrs Rebekah Jukes‐Jones and Dr Claudia Langlais for all the time and effort put into the mass spectrometry aspect of this project, and the feedback and discussions which followed. I’m grateful also to members of the Willis group for their contributions, in particular Dr Mark Stoneley for his method development input in the early stages and Dr Ruth Spriggs for help with analysis. Finally, I would like to thank my parents ‐ Chris and Sharon, my sisters, my partner Nik and all the friends near and far, who may not have understood exactly what I did but understood that I loved it, and supported me throughout. 3 Contents 1 INTRODUCTION.............................................................................................................. 18 1.1 GENES TO PROTEINS: CONTROL OF GENE EXPRESSION .............................................................. 18 1.2 EUKARYOTIC TRANSLATION: AN OVERVIEW ........................................................................... 19 1.2.1 RIBOSOMES ........................................................................................................................ 19 1.2.2 TRANSLATIONAL INITIATION ................................................................................................... 23 1.2.3 TRANSLATIONAL ELONGATION................................................................................................ 29 1.2.4 TERMINATION OF TRANSLATION ............................................................................................. 30 1.2.5 REGULATION OF TRANSLATIONAL CONTROL .............................................................................. 31 1.3 RNA BINDING PROTEINS ................................................................................................... 31 1.3.1 RNA BINDING DOMAINS AND POST‐TRANSCRIPTIONAL MODIFICATIONS ........................................ 32 1.3.2 RNA BINDING PROTEINS INVOLVED IN RNA PROCESSING AND SPLICING ........................................ 33 1.3.3 RNA BINDING PROTEINS, SEQUESTRATION AND SPATIAL REGULATION ........................................... 34 1.3.4 RNA BINDING PROTEINS AND TRANSLATIONAL CONTROL ............................................................ 35 1.3.5 RNA BINDING PROTEINS AND METABOLISM ............................................................................. 35 1.3.6 RNA BINDING PROTEINS AND RNA DECAY ............................................................................... 36 1.3.7 DISEASE STATES ARISING FROM RNA BINDING PROTEIN DYSREGULATION ...................................... 37 1.3.8 METHODOLOGIES FOR IDENTIFICATION OF CELLULAR RNA BINDING PROTEINS ............................... 37 1.4 SIGNALLING CASCADES AND SIGNAL TRANSDUCTION ................................................................ 40 1.4.1 KINASES VS PHOSPHATASES ................................................................................................... 41 1.4.2 THE MAMMALIAN TARGET OF RAPAMYCIN SIGNALLING .............................................................. 41 1.4.3 MTOR ACTIVATION AND SIGNAL TRANSDUCTION ...................................................................... 44 1.4.4 MTOR REGULATION OF TRANSLATION .................................................................................... 45 1.4.5 MTOR AND METABOLISM ..................................................................................................... 50 1.4.6 MTOR AND AUTOPHAGY....................................................................................................... 50 1.4.7 MTOR SIGNALLING AND DISEASE ............................................................................................ 51 1.4.8 DEVELOPMENT OF KINASE INHIBITORS ..................................................................................... 51 1.4.9 THERAPEUTIC RELEVANCE OF MTOR INHIBITORS ....................................................................... 52 1.5 PROJECT AIMS ................................................................................................................ 53 2. MATERIALS AND METHODS .......................................................................................... 56 2.1. BUFFERS AND SOLUTIONS ................................................................................................ 56 2.2. CELL CULTURE TECHNIQUES ............................................................................................... 57 2.2.1. GENERAL CELL CULTURE ....................................................................................................... 57 2.2.2. SIRNA TRANSFECTIONS ........................................................................................................ 57 2.2.3. GROWTH CURVES AND CELL COUNTING .................................................................................. 58 2.2.4. FACS ANALYSIS .................................................................................................................. 58 2.2.5. MICROSCOPY TO ASSESS CELL MORPHOLOGY ........................................................................... 59 2.3. RNA TECHNIQUES .......................................................................................................... 59 2.3.1. RNA EXTRACTION ............................................................................................................... 59 2.3.2. RT‐QPCR WITH SYBRGREEN ................................................................................................ 60 4 2.3.3. SUCROSE GRADIENT POLYSOME PROFILING ............................................................................ 61 2.4. PROTEIN TECHNIQUES ..................................................................................................... 63 2.4.1. SONICATION AND NORMALISATION OF PROTEIN BY BRADFORD .................................................. 63 2.4.3. WESTERN BLOTTING ........................................................................................................... 65 2.4.4. WHOLE CELL RNA BINDING PROTEIN INTERACTOME CAPTURE .................................................. 67 2.4.5. MASS SPECTROMETRY ......................................................................................................... 69 2.4.6. MASS SPECTROMETRY ANALYSIS ........................................................................................... 70 2.4.7. FLAG‐TAGGED PCMV‐LARP1
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