Abstract:
N6-Methyladenosine (m6A) is a prevalent post-transcriptional modification in eukaryotic messenger RNA. Two cancer-linked human Fe(II) and
2-oxoglutarate (2OG)-dependent oxygenases, the fat mass and obesity associated-protein (FTO), and AlkB human homolog 5 (ALKBH5) catalyse
m6A methyl group oxidation. While ALKBH5 has consistently been reported to catalyse m6A demethylation, there are conflicting reports con-
cerning the FTO products. We report studies using mass spectrometry and nuclear magnetic resonance comparing products of FTO, ALKBH5,
and DNA damage repair demethylases (human ALKBH2 and ALKBH3 and bacterial AlkB, using m1A single-stranded DNA substrates). The results
with m6A-containing single-stranded RNA (ssRNA) and N6,2
-O-dimethyladenosine adjacent to the 5 m7G triphosphate cap ssRNA substrates
imply that the predominant FTO product is N6-hydroxymethyladenosine, either with or without methylation on the substrate ribose 2
-hydroxyl
group. The nascent hemiaminal product undergoes relatively slow non-enzyme catalysed fragmentation giving adenosine/formaldehyde. The
other four 2OG-dependent oxygenases tested, including ALKBH5, produce demethylated bases as the predominant products. The results imply
that, at least in isolated form, FTO preferentially acts as a hydroxylase, producing a hemiaminal product, rather than a demethylase, distinguishing
it from ALKBH5. They highlight a need for investigations into the roles of hemiaminal-type modifications to nucleic acids, in both healthy biology
and disease.
