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Primary Supervisor: Prof Sarah Blagden

Second Supervisor: Prof Kathryn Lilley, Dept Biochemistry, University of Cambridge 

Project Overview:

Aberrant cellular metabolism is one of cancer’s key defining hallmarks. Cancer cells are uniquely capable of simultaneously activating glycolysis and aerobic respiration to overcome environmental hypoxia and nutrient starvation. This metabolic plasticity enables the cells to develop chemotherapy and radiotherapy resistance; hence therapeutically targeting metabolism is a recognised anticancer strategy. However, the biology underpinning plasticity has yet to be fully characterised. Following our discovery that the La-related protein (LARP) family has a central role within it (1), the purpose of this project is to explore this regulatory mechanism in more detail and identify new targets and biomarkers that can contribute to cancer treatments of the future.    We have discovered that the evolutionarily-ancient RNA binding proteins LARP1 and LARP1B link growth factor signalling to the expression of networks of metabolic enzymes. LARP1 is frequently highly expressed in cancer where it promotes tumorigenesis and cancer progression by enabling cells to adapt to nutrient starvation and modulate the local tumour immune microenvironment (2). LARP1 and 1B carry two major RNA binding domains and are extensively phosphorylated by upstream kinases, including mTORC1, CK2 and AKT1 whereupon they bind and stabilise mRNAs encoding multiple cellular metabolic enzymes. In this project, we will establish how phosphorylation of LARP1 and LARP1B contributes to mRNA selectivity and tumorigenesis.   To address this, we will utilise state-of-the-art technologies developed at the Universities of Oxford and Cambridge including mass-spectrometry based phosphoproteomics combined with an unbiased RNA interactome capture method, OOPS, to determine the role of phosphorylated amino acids of LARP1 in RNA binding (3). We will also apply individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) to determine the identity of the RNA molecules coordinated by LARP1. Together, these approaches will shed light on  the “phosphorylation code” for LARP1 and identify protein–RNA crosslink sites on a genome-wide scale. We have already generated a wide range of LARP1 knockout cells and cells expressing LARP1 phospho-mutants which, alongside colorectal organoids, will be used to investigate the functions of specific LARP1 phospho-sites.   

Training Opportunities:

We will train the student in several cutting-edge techniques for investigating protein-RNA interactions and cancer biology including: iCLIP and Ribo-STAMP (Surveying Targets by APOBEC-Mediated Profiling) or orthogonal organic phase separation (OOPS).   Preparation and analysis of samples for mass-spectrometry based phosphoproteomics. CRISPR-Cas9 for generation of knockout and phosphomutant cell lines and organoids Cancer organoid maintenance and model systems Immunofluorescence microscopy and flow cytometry

Relevant Publications:

Chettle, J., Dedeic, Z., Fischer, R., Vendrell, I., Campo, L., Easton, A., Browne, M., Morris, J., Schwenzer, H., Lascaux, P. and Gijsbers, R., 2022. LARP1 regulates metabolism and mTORC1 activity in cancer. bioRxiv, pp.2022-09.

Queiroz, R.M., Smith, T., Villanueva, E., Marti-Solano, M., Monti, M., Pizzinga, M., Mirea, D.M., Ramakrishna, M., Harvey, R.F., Dezi, V. and Thomas, G.H., 2019. Comprehensive identification of RNA–protein interactions in any organism using orthogonal organic phase separation (OOPS). Nature biotechnology37(2), pp.169-178.

Smith, E.M., Benbahouche, N.E.H., Morris, K., Wilczynska, A., Gillen, S., Schmidt, T., Meijer, H.A., Jukes-Jones, R., Cain, K., Jones, C. and Stoneley, M., 2021. The mTOR regulated RNA-binding protein LARP1 requires PABPC1 for guided mRNA interaction. Nucleic acids research49(1), pp.458-478.