FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA

N-6-methyladenosine is a prevalent internal modification in messenger RNA and non-coding RNA affecting various cellular pathways. Here we report the discovery of two additional modifications, N-6-hydroxymethyladenosine (hm(6)A) and N-6-formyladenosine (f(6)A), in mammalian messenger RNA. We show that Fe-II-and alpha-ketoglutarate-dependent fat mass and obesity-associated (FTO) protein oxidize N-6-methyladenosine to generate N-6-hydroxymethyladenosine as an intermediate modification, and N-6-formyladenosine as a further oxidized product. N-6-hydroxymethyladenosine and N-6-formyladenosine have half-life times of similar to 3 h in aqueous solution under physiological relevant conditions, and are present in isolated messenger RNA from human cells as well as mouse tissues. These previously unknown modifications derived from the prevalent N-6-methyladenosine in messenger RNA, formed through oxidative RNA demethylation, may dynamically modulate RNA-protein interactions to affect gene expression regulation.