Characterisation of the RNA processing factor CIP29 (HCC-1/SARNP) and its involvement in cell proliferation and genome stability

Edge, Giselle and Taylor, Elaine and Lindsay, Howard (2024) Characterisation of the RNA processing factor CIP29 (HCC-1/SARNP) and its involvement in cell proliferation and genome stability. PhD thesis, Lancaster University.

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Abstract

Cancer has become a leading cause of death worldwide, originating when cells incur genetic damage and altered gene expression that leads to dysregulated proliferation and other cancer hallmarks. Mechanisms that help to maintain genome integrity and the correct expression of this genetic information, such as effective DNA repair, accurate chromosome segregation and regulated gene expression are therefore fundamentally important in curbing the development and progression of cancer. Correct gene expression relies on both transcriptional regulation and correct processing, export and translation of mRNA transcripts, the mediators between the genetic instructions and protein synthesis. The journey an mRNA transcript undergoes during RNA processing can affect its stability and behaviour which in turn, can profoundly influence gene expression and cellular behaviour. An RNA processing factor of interest CIP29, is often upregulated in cancer cells and may aid cancer cell development and growth. Although implicated in RNA processing and export, the precise function of CIP29 has not been fully characterized. To gain a greater understanding of the activities of CIP29 we attempted to identify and validate CIP29-interacting factors and to phenotypically characterise cells in which CIP29 had been abolished through CRISPR-Cas9 gene editing. Upon examination, only proteins that had been previously implemented within mRNA export were shown to reliably associate with the CIP29 protein, suggesting that the influence of CIP29 within the cell is achieved through its role as an mRNA exporter. CIP29 knockout cells showed no evidence of genetic damage but displayed a profound defect in cell cycle progression. We have characterised this cell proliferation problem as relating to defective chromosome segregation and cell division. These defects were not observed once the CIP29 protein was reintroduced through CIP29-rescue cell lines, suggesting that the mitotic defects observed were because of the loss of CIP29. To further obtain an understanding of the functional domains and regions of interest within the CIP29 protein, its molecular structure was examined through the development of truncated mutants which were then subjected to phenotypic analysis. The results obtained suggested that the C-terminal end of the protein was significant within the mitotic defects observed, as truncations without this region displayed a phenotype that was like or exceeded the CIP29-knockout. Overall, the results obtained suggest that the CIP29 protein is fundamental in the successful completion of the cell cycle, which may be due to its role in the processing and export of mRNA transcripts for key mitotic proteins.