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BRCA1 is a breast cancer susceptibility gene that is down-regulated in the majority of cases of sporadic breast cancer. Accordingly, there is considerable interest in the mechanisms that regulate normal expression of BRCA1, with a view to elucidating how this could be disrupted in breast cancer. In tumours with reduced BRCA1 protein expression, there can be a concomitant reduction in mRNA level to variable degrees, or no change in mRNA level, suggesting that disruption of multiple different regulatory processes may contribute to BRCA1 down-regulation. Despite this, efforts to date have chiefly focussed on transcriptional and epigenetic regulation of the gene, whilst post-transcriptional processes that regulate the dynamics of the BRCA1 transcript, such as decay, localisation and translation efficiency, are poorly understood. Post-transcriptional regulatory pathways are critical for sustaining normal cellular physiology, as evidenced by many examples where disruption of these processes results in disease, including cancer. Regulation of gene expression at this level is often mediated by RNA-binding proteins that recognise specific cis-acting sequence motifs in the untranslated regions (UTRs) of certain messenger RNAs, and recruit, or shield them from macromolecular complexes involved in RNA metabolism, such as the translation apparatus, exosome, and subcellular transport particles. This thesis is centred on investigating post-transcriptional regulation of BRCA1. Others have shown that expression of the transcript and protein is regulated throughout the mammalian cell division cycle. Results presented in this thesis suggest that changes in mRNA stability may contribute to cell cycle-dependent expression of BRCA1, and therefore that post-transcriptional regulation of BRCA1 is a biologically-relevant phenomenon. To begin to address the molecular mechanisms involved in regulation of BRCA1 mRNA decay, and possibly other post-transcriptional regulatory processes, the 3UTR of BRCA1, which had not been previously characterised, was analysed for functional regulatory motifs using a combination of bioinformatics, reporter assays and RNA-proteinbinding analysis. An evolutionarily-conserved 3UTR subsequence was identified which contains sequence elements capable of regulating reporter activity, and forming complexes with multiple proteins from human epithelial cell lines. Some of these elements have been previously characterised in the context of other genes, including a Hu-antigen R (HuR)-binding motif, adenosine-uridine (AU)- rich sequences and a differentiation control element (DICE). Experiments were also conducted to determine the identities of the RNA-binding proteins detected using an RNA probe containing the 3UTR elements. A preliminary screen of a small group of RNA-binding proteins with previously-characterised roles in 3UTR-mediated post-transcriptional gene regulation identified HuR as a negative regulator of BRCA1 protein expression. Interestingly, HuR is over-expressed in breast cancer. Evidence presented in this thesis suggests that the mechanism of HuR-mediated down-regulation of BRCA1 involves direct binding of HuR to the BRCA1 3UTR, and no changes to mRNA stability or abundance. Finally, proteomics-based analysis of protein extracts enriched with BRCA1 3UTR RNA-binding proteins yielded several interesting candidates with previously-reported RNA-binding and/or post-transcriptional regulatory activities, including Far upstream element-binding protein 1 (FBP1), Glyceraldehyde-3-phosphate dehydrogenase (GAPD) and Heat-shock protein 27 (HSP27). This thesis addresses a clear deficiency in the literature concerning regulation of BRCA1, and contributes to our general understanding of the molecular mechanisms controlling gene expression in mammalian cells. Additionally, the finding that RNA-binding proteins that are over-expressed in breast cancer can negatively regulate BRCA1 expression constitutes important groundwork for identifying potential novel breast cancer therapeutics in the future. |
