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A gene deletion strategy to identify the function of a non-coding RNA in the eukaryotic genome using the model organism Saccharomyces cerevisiae
[Thesis]. Manchester, UK: The University of Manchester; 2015.
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Abstract
Non-coding RNA (nc-RNA) plays an important role in biological processes. To understand a non-coding RNA function, we constructed twelve molecular bar-coded deletion mutants in Saccharomyces cerevisiae including five snRNAs, three RNAs of unknown function (RUFs), TLC1, SCR1, NME1 and RPR1. Nine of the twelve genes were found to be essential. RUF20 was particularly interesting as it was essential and overlaps the 3’ untranslated region (UTR) of SEC4, a GTPase essential for vesicle-mediated exocytic secretion and autophagy. Shorter RUF20 deletions and SEC4 plasmid complementation in RUF20 knock-out strains concluded that RUF20 essentiality was derived from overlap with the SEC4 3’ UTR. The SEC4 3’ UTR is required for localisation of SEC4 mRNA to bud tips and the cell membrane. To investigate the molecular mechanisms of how RUF20 regulates SEC4 3’ UTR formation or SEC4 function, RUF20 expression was turned off by using the TetO7 promoter system. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were performed to determine mRNA abundance for the targeted genes (RUF20 and SEC4). It was found that SEC4 mRNA expression was decreased when RUF20 mRNA expression was suppressed. SEC4 3’ UTR formation was checked by RT-PCR primer walking, which indicated that SEC4 3’ UTR processing was affected and not formed when RUF20 expression was inhibited. To investigate the localisation of RUF20 and SEC4 in the presence/absence of RUF20 RNA, fluorescence in situ hybridisation (FISH) was performed and it was found that RUF20 RNA displayed a similar pattern of localisation with SEC4 mRNA and there was a mislocalisation of SEC4 mRNA if RUF20 RNA was not expressed. We have identified a novel role for a non-coding RNA suggesting that RUF20 is required for SEC4 mRNA expression and influences the 3’ end formation and SEC4 mRNA localisation.