Investigation of RNA binding proteins regulated by mTOR

• Main Author: Morris, Katherine Louise
• Other Authors: Bushell, Martin ; Cain, Kelvin
• Published: University of Leicester 2017
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.713377

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.