Credit: Brian Harris/Alamy

The discovery that reversible mRNA modifications provide a tuneable layer regulating gene expression has galvanized the field of epitranscriptomics. Now, Mauer et al. report that one of the most prevalent modified bases, N6,2′-O-dimethyladenosine (m6Am), found in 30% of mRNAs, is a dynamic and reversible modification that confers mRNA stability.

In contrast to internal base modifications such as N6-methyladenosine (m6A), m6Am is found at the 5′ end of mRNAs, when the first nucleotide following the 5′ cap is a 2′-O-methyladenosine that is modified by additional N6-methylation. Although the prevalence of m6Am had been known for some time, its function remained elusive.

The demethylase FTO was previously linked to the demethylation of m6A, but the team suspected that FTO-regulated peaks in transcriptome-wide maps might reflect m6Am rather than m6A. The authors incubated a synthetic oligonucleotide, in which m6Am was positioned following a 5′ cap, with FTO, which readily demethylated m6Am, as assessed by high-performance liquid chromatography. Competition with another oligonucleotide containing m6A showed that FTO had higher activity towards m6Am than m6A, suggesting that m6Am is the preferred substrate of this enzyme. This finding was confirmed in vivo using HEK293T cells transfected with a tagged FTO. Transfection led to significantly reduced levels of m6Am, which could be decreased further by inducing the cytosolic translocation of FTO. By contrast, knockdown or knockout of FTO expression increased the amount of m6Am in vivo, but had no effect on m6A levels.

Interestingly, mRNAs beginning with m6Am were found to be substantially more stable, showing an average increase in half-life of 2.5 h, and exhibited higher transcript levels than mRNAs starting with any other nucleotide. Manipulation of m6Am levels through FTO overexpression or knockdown indicated that demethylation of this RNA modification reduces mRNA stability, whereas increasing m6Am levels enhances the stability of mRNAs beginning with this modified nucleotide.

Given that mRNA degradation often involves decapping, the authors set out to determine whether this process is affected by m6Am. In vitro experiments found that RNAs with a 5′ cap followed by m6Am exhibited significantly reduced decapping mediated by the mRNA-decapping enzyme DCP2. Furthermore, levels of m6Am mRNAs did not change as dramatically as those of other mRNAs in DCP2-deficient HEK293T cells compared with controls, which suggests that m6Am confers protection from DCP2-mediated degradation.

The findings raise a number of questions that the authors hope to address going forward. “Many aspects of extended cap methylation are yet to be explored,” says lead author Jan Mauer (Weill Cornell Medicine, Cornell University). “For example, how does m6Am affect ribosome binding? Or what physiological stimuli activate demethylation of m6Am?” The preference of FTO for m6Am also raises doubts over some of the previously established dynamics of the m6A modification.

Overall, the study clearly demonstrates that the location of modified nucleotides along the mRNA and the exact combination of modifications on the nucleotide all play a crucial part in modulating epitranscriptome function. “Our findings suggest that the mRNA cap does not simply serve as a docking platform for the translation machinery, but can actually carry information encoded by the modification state of the first nucleotide,” concludes Mauer.