Phd in GENETICS AND MOLECULAR BIOLOGY

Thesis title: Deciphering the role of N6-Methyladenosine in the metabolism of circular RNAs

Circular RNAs (circRNAs) are a class of covalently closed single-stranded RNA molecules produced through a back-splicing reaction, where a downstream 5’ splice donor site is joined to an upstream 3’ splice acceptor site. Several studies demonstrated their functional role in many physiological and pathological processes; nevertheless, to date little is known about the cellular mechanisms regulating circRNA biogenesis and function. N6-Methyladenosine (m6A) is the most abundant internal modification of coding and non-coding RNA polymerase II transcripts, with a well-known contribution to different processes controlling RNA metabolism. Conversely, the role of m6A in the regulation of circRNAs is yet to be addressed. We started our investigation using circ-ZNF609, a methylated circRNA, as a study case. We showed that the m6A writer METTL3 and the nuclear reader YTHDC1 are required to direct ZNF609 precursor towards the back-splicing reaction, without competition with the production of the linear isoform. This feature is shared with a larger group of circRNAs in HeLa cells, since we found a significant direct correlation among METTL3 requirement, YTHDC1 binding and the ability of m6A exons to undergo back-splicing. We further characterized m6A-dependent regulation in the pathological context of rhabdomyosarcoma (RMS), where circ-ZNF609 levels were showed to correlate with YTHDC1 expression. We demonstrated that circularization is enhanced in two different model cell lines of RMS when compared to human wild-type myoblasts. In line with a possible m6A-driven increase of circRNA expression in RMS, we showed that protein levels of METTL3 and its co-factor METTL14, as well as YTHDC1, are higher in RMS. We were also able to establish a dependency of RMS growth on the presence of m6A per se through the action of METTL3. During our investigation, we identified a novel co-factor of the m6A regulatory machinery, DDX5. We showed that DDX5 expression correlates with the one of YTHDC1 in human and mouse and that the two proteins are able to physically interact with each other. Moreover, YTHDC1 and DDX5 seem to participate in common pathways regulating RMS growth. We showed that circRNA levels in RMS are dependent on the action of YTHDC1 and even more of DDX5, which is a novel regulator of the back-splicing reaction. A significant class of circRNAs is regulated by both proteins; among them, we found mostly circRNAs up-regulated in RMS when compared with wild-type myoblasts. Therefore, we identified new bona fide oncogenic circRNAs whose higher levels in RMS are sustained by combined action of m6A mediated by the action of YTHDC1 and DDX5.