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Abstract
RNA was originally considered to be a molecule whose primary function was to encode proteins. When it was discovered during the human genome project that the number of protein coding genes was radically lower than previously estimated, the idea was posited that non-coding RNA (ncRNA) could contribute to increasing complexity and account for the observed similarity of number of protein-coding genes between humans and other less complex organisms. Over the last decade, experimental observations have been used to categorise non-coding RNAs by function, which increasingly points towards them being involved in performing a role in regulating the transcriptional and epigenetic states of gene expression.
In this thesis, a number of ncRNA species have been studied to ascertain a greater understanding of the mechanisms involved in transcriptional and epigenetic regulation. I examine short antisense RNA directed transcriptional gene silencing of Periostin, resulting in a loss of cellular metastasis. Periostin is involved in altering the extracellular matrix to favour increased invasive potential. I investigate the mechanisms involved in the complex PTEN regulatory network, involving RNA:RNA hybrid interactions between an lncRNA pseudogene transcript and a 5’-UTR ncRNA transcript the PTEN promoter which regulate gene expression of PTEN via transcriptional and epigenetic regulation. I also studied the deamination DNA repair pathways, resulting in the arisal of cytosine to thymine SNP mutations at loci targeted by shRNAs, antisense lncRNAs, and small antisense RNAs when MBD4 and SMUG repair enzymes have been repressed. Collectively, these data suggest a far more complex role for ncRNAs in the cell has been understood previously, as they appear to play a large part in directing epigenetic regulation and modification of the human genome.