Non-coding RNAs (ncRNAs) are a subset of RNAs that do not code for protein. They are divided into a number of different groups based on their function and targets. Small nucleolar RNAs (snoRNAs) are ncRNAs that have long been known to function as guides for ribosomal RNA (rRNA) modifying enzymes. They are classified into two major groups: box C/D snoRNAs and box H/ACA snoRNAs. Most box C/D snoRNAs direct the 2'-O-methylation of rRNA substrates, but some lack known targets and are therefore termed 'orphan snoRNAs'. Studies have implicated orphan snoRNAs in pre-mRNA processing and stability, but the functional consequence of snoRNA binding to mRNAs has not been fully determined. Saccharomyces cerevisiae had two orphan snoRNAs, snR4 and snR45, with no known function in ribosome synthesis. This project aimed to determine the targets of these snoRNAs, and investigate the effects of snoRNA binding to non-canonical target RNAs, as well as the underlying mechanism. Synthetic gene array screens with deletions of the SNR4 and SNR45 genes identified multiple positive and negative genetic interactions. In particular, deletion of either snoRNA gene was synthetic-lethal with mutation of the snoRNA-associated methyltransferase, Nop1 (Fibrillarin in humans), demonstrating that both have important functions. CLASH analyses of RNA-RNA interactions showed that these snoRNAs bind multiple mRNAs, while RNA sequencing and RT-qPCR revealed that snoRNA deletion altered mRNA abundance. Both orphan snoRNAs were well conserved between fungi, with a region of high conservation indicating a potential binding site. Associations were identified between snR4 and snR45 and multiple sequences within rRNA, including two recently identified sites of 18S rRNA acetylation. Work elsewhere showed that snR4 and snR45 function as guides for the acetyltransferase Kre33 using the region of high conservation, removing their 'orphan' status. Orphan snoRNAs have been implicated in human diseases, such as Prader Willi Syndrome and cancers. The work discussed in this thesis helps to elucidate the RNA interactions of yeast orphan snoRNAs. It has provided a greater understanding of the mechanisms involved, and may inform future work in combatting human disease.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:763945 |
| Date | January 2018 |
| Creators | Peters, Rosie Elizabeth |
| Contributors | Tollervey, David ; Beggs, Jean |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/33123 |
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