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5-methylcytidine has a complex, context-dependent role in RNA

Shafik, Andrew Mark

Description

Ribonucleic acid (RNA) metabolism processes and function are affected by specific RNA sequence motifs, the ability of RNA to form secondary structure and assemble into ribonucleoprotein (RNP) complexes (Dandekar & Bengert, 2002). As these aspects are likely to be affected by nucleoside modifications, it is important to document the extent and function of post-transcriptional modifications present in these molecules. Thus, there is an increasing focus on...[Show more]

dc.contributor.authorShafik, Andrew Mark
dc.date.accessioned2017-06-27T00:14:19Z
dc.date.available2017-06-27T00:14:19Z
dc.identifier.otherb44884023
dc.identifier.urihttp://hdl.handle.net/1885/118266
dc.description.abstractRibonucleic acid (RNA) metabolism processes and function are affected by specific RNA sequence motifs, the ability of RNA to form secondary structure and assemble into ribonucleoprotein (RNP) complexes (Dandekar & Bengert, 2002). As these aspects are likely to be affected by nucleoside modifications, it is important to document the extent and function of post-transcriptional modifications present in these molecules. Thus, there is an increasing focus on exploring the incidence and biological relevance of post-transcriptional marks in RNA. This thesis is aimed at defining a role for the post-transcriptional modification, 5- methylcytidine (m5C) particularly in mRNAs. Bisulfite treatment, which allows for the nucleotide-specific identification of m5C, coupled with next generation sequencing allowed for the detection of m5C sites transcriptome-wide at single-nucleotide resolution. This revealed candidate m5C sites in many RNA biotypes including both non-coding and coding RNA in cervical cancer cells (HeLa). In mRNAs, m5C candidate sites were observed in both untranslated regions and in the coding sequence, suggesting multiple roles for m5C. Global analyses revealed features of the m5C modification in nuclear-encoded mRNAs. m5C sites were shown to be relatively enriched in the 5′ UTR, to be associated with high GC content, low minimum free energy structures, and weakly translated mRNAs. Furthermore, experimental and bioinformatic approaches suggested a role for m5C in regulating mRNA translation by recruiting specific m5C-binding proteins, which were identified through a RNA bait pulldown approach. m5C was also suggested to function in stabilising target mRNA. Bioinformatic and experimental approaches indicated m5C might physically block the destabilising effect of Argonaute 2/microRNA binding. In this way, m5C would protect the transcript from degradation. Also, further work identified the m5C modification in mitochondrial (mt) rRNA, where a function of m5C is discussed, in novel structural positions in mt-tRNAs, but not in mt- mRNAs. Altogether, this thesis suggests that m5C has a complex, context-dependent role in RNA.
dc.language.isoen
dc.subject5-methylcytosine
dc.subjectwriters
dc.subjectreaders
dc.subjecterasers
dc.subjectbisulfite
dc.subjectNGS
dc.subjecttranscriptome-wide
dc.title5-methylcytidine has a complex, context-dependent role in RNA
dc.typeThesis (PhD)
local.contributor.supervisorPreiss, Thomas
local.contributor.supervisorcontactthomas.preiss@anu.edu.au
dcterms.valid2017
local.description.notesthe author deposited 27/06/2017
local.type.degreeDoctor of Philosophy (PhD)
dc.date.issued2016
local.contributor.affiliationDepartment of Genome Sciences, The John Curtin School of Medical Research (JCSMR), The Australian National University
local.identifier.doi10.25911/5d6fa1dd8400d
local.mintdoimint
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