Abstract

N6-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, N6-hydroxymethyladenosine (hm6A) and N6-formyladenosine (f6A), in mammalian messenger RNA. We show that FeII- and α-ketoglutarate-dependent fat mass and obesity-associated (FTO) protein oxidize N6-methyladenosine to generate N6-hydroxymethyladenosine as an intermediate modification, and N6-formyladenosine as a further oxidized product. N6-hydroxymethyladenosine and N6-formyladenosine have half-life times of ~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 N6-methyladenosine in messenger RNA, formed through oxidative RNA demethylation, may dynamically modulate RNA–protein interactions to affect gene expression regulation. Internal modifications in mRNA and non-coding RNA are necessary for modulating various intracellular signalling pathways. In this study, the authors report novel modifications resulting from oxidative RNA demethylation, which regulate RNA–protein interactions affecting gene expression.