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Title: Cellular studies on the role of OGFOD1, a 2-oxoglutarate-dependent dioxygenase
Author: Attwood, Martin
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2017
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2-Oxoglutarate (2-OG) and iron (Fe2+) dependent Oxygenase Domain containing protein 1 (OGFOD1) is an evolutionarily conserved prolyl hydroxylase enzyme in mammals, invertebrates (Sud1 in D. melanogaster) and yeast (TPA1 in S. cerevisiae and Ofd1 in S. pombe). This enzyme modifies a residue corresponding to Proline-62 in humans of the small ribosome protein S23 (RPS23) that occupies a critical site within the decoding centre of the eukaryotic ribosome. A number of cellular phenotypes have been reported following OGFOD1 inactivation, including altered ribosome decoding, activation of translational stress pathways and reduced proliferation. In addition, studies in S. pombe have assigned a novel signalling function for Ofd1 in the regulation of sterol response genes via the Sre1 transcription factor. Collectively, these studies indicated functions for OGFOD1 in both translation and transcription. However, it was not known whether these functions are conserved in mammalian cells or if they are linked. To inform on OGFOD1-dependent cellular phenotypes, OGFOD1 defective cells were generated by genome editing using the CRISPR methodology. Clones re-expressing the OGFOD1 transgene under the control of a doxycycline regulable promoter were subsequently derived, facilitating comparison of OGFOD1 status within an isogenic setting. These cells were validated by the generation and use of antibodies specific to the modification site (Pro-62) in RPS23. Surprisingly, genetic inactivation of OGFOD1 did not mimic the reported phenotypes observed by RNA interference. To improve understanding of OGFOD1 function, immunoprecipitations of ectopic and endogenous OGFOD1-containing protein complexes were analysed by proteomic methods. The transcriptional regulator, Thanatos associated protein 11 (THAP11) was identified as a novel high stoichiometry OGFOD1-interacting protein across a range of cell lines. Analysis of the THAP11 transcriptional programme in SW620 cells defined a repressive action of OGFOD1 overexpression on THAP11 DNA binding and target gene expression. Reciprocal regulation of THAP11 signalling was not associated with OGFOD1 knockdown or genetic inactivation suggesting that THAP11 is not limiting in the steady state. Protein interaction studies revealed a direct association between the two proteins that was mediated by a region of THAP11 that shares sequence homology to the Ofd1-binding domain of Sre1. This, together with the observation that THAP11 forms a discrete association with the translational machinery, raises the interesting possibility that OGFOD1 performs an analogous regulatory function in mammalian cells to that described in S. pombe. These results provide a focus for future work dissecting the interrelationship between OGFOD1-dependent effects on transcription and translation.
Supervisor: Cockman, Matthew ; Ratcliffe, Peter Sponsor: Ludwig Institute for Cancer Research
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Ribosomal hydroxylation ; Ribosomopathy ; OGFOD1 ; 2-oxoglutarate-dependent dioxygenase ; RPS23