FTO-Mediated Formation of N(6)-Hydroxymethyladenosine and N(6)-Formyladenosine in Mammalian RNA

N(6)-methyladenosine (m(6)A) is a prevalent internal modification in mRNA 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 mRNA. We show that Fe(II)- and α-ketoglutarate (α-KG)-dependent fat mass and obesity associated (FTO) protein oxidizes m(6)A to generates hm(6)A as an intermediate modification and f(6)A as a further oxidized product. hm(6)A and f(6)A have half-life times of ~3 h in aqueous solution under physiological relevant conditions, and are present in isolated mRNA from human cells as well as mouse tissues. These previously unknown modifications derived from the prevalent m(6)A in mRNA, formed through oxidative RNA demethylation, may dynamically modulate RNA-protein interactions to affect gene expression regulation.