5-methylcytidine has a complex, context-dependent role in RNA
Date
2016
Authors
Shafik, Andrew Mark
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Abstract
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 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.
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Keywords
5-methylcytosine, writers, readers, erasers, bisulfite, NGS, transcriptome-wide
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Thesis (PhD)
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