Discovery of N6-methyladenosine on R-loops and elucidating its role in their regulation
Publication Date
October 15, 2020
Creator
Abstract
Amongst the hundreds of known chemical modification of RNA, N6- methyladenosine (m6A) is the most abundant internal modification found on messenger RNAs. Despite this, the biological function of m6A remained largely enigmatic, in part due to earlier studies suggesting its static nature. The discovery of enzymes capable of reversing m6A demonstrated its dynamic nature. This was complemented by development of methodologies that allowed transcriptome-wide analysis of m6A distribution in eukaryotic organisms from yeast to human, with the subsequent implication of m6A in almost all steps of RNA metabolism. Meanwhile, N6-methyldeoxyadenine (6mA), its DNA counterpart that, for decades, was thought to be restricted to prokaryotic genomes and single cell eukaryotes, has recently been detected in multi-cellular eukaryotes C. elegans, Drosophila and green algae. However, due to contradictory reports regarding its detection, the presence and potential biological function of 6mA in mammalian genomes was unclear. In search of 6mA in human pluripotent stem cell genome, this thesis corroborated other studies in failing to detect 6mA in human cells. Nonetheless, this thesis describes the discovery of m6A presence on the RNA component of R-loops, the three-stranded nucleic acid structures consisting of RNA:DNA hybrid and a displaced single stranded DNA, that can confer vulnerability to genotoxic attacks. Through analysis of cell-cycle resolved R-loops, this thesis reveals the dynamic nature of m6A modified RNA:DNA hybrids, notably, with their higher accumulation in S and G2/M cell cycle phases compared to G0/G1 cells. Moreover, this thesis uncovers direct interaction of the m6A reader, YTHDF2 with R-loops, both in vitro and in vivo. Finally, this thesis uncovers a role for both m6A and YTHDF2 in regulating the stability of R-loops, as depletion of either the m6A writer, METTL3 or YTHDF2 resulted in genome-wide R-loops accumulation. Moreover, persistent loss of YTHDF2 led to cell growth retardation, increased accumulation of RNA:DNA hybrids and extensive DNA damage in Rloop-containing loci. Taken together, the findings of this thesis uncover a role for m6A and YTHDF2 in safeguarding the genome by limiting toxic accumulation of R-loops.
Item Type
ethesis
Thesis Type
PhD
Supervisors
Subjects (LC)
Associated Schools / Departments
School of Medicine
eprints ID
61382
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Abdulkadir Abakir PhD Thesis_The Discovery of N6-methyladenosine on R-loops and elucidating its role in their regulation.pdf
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