Biologically derived, chemically-modified RNA molecules represent a ubiquitously present but poorly understood area of molecular biology. Despite this substantive gap in our understanding RNA methyltransferase (RNMT) enzymes, which catalyse the formation of RNA 5mC, are incredibly highly conserved, while disrupting m6A metabolism leads to early embryo lethality. This indicates that these modifications perform critical, but poorly characterised, biological functions.
Knockout experiments and clinical pathologies guided the work of this thesis in which RNA methylation was analysed in the context of epigenetic reprogramming and devlopment more broadly. LC-MS/MS-based quantification techniques were developed to achieve unparalleled sensitivity and applied to in vivo and in vitro murine models of epigenetic reprogramming and developmental progression through pluripotency. While an intrinsic link between DNA 5mdC methylation and RNA 5mC methylation was refuted, 5mC changes dynamically along a naive-primed pluripptent axis, in line with the redistribution of NOP2 and NSUN2 within the cell. While these RNMTs are strongly linked in bringing about these changes in 5mC abundance, increased expression of NSUN2 in the trophectoderm at the blastocyst stage uncovers a potential lineage specific role for this enzyme.
This study reveals hitherto undiscovered connections between RNA methylation and the developmental transitions into and out of pluripotency. Better understanding of these molecular processes may ultimately lead into more refined and effective approaches to regenerative medicine.