Reversible methylation of m⁶A_m in the 5' cap controls mRNA stability

Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5' end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N⁶,2'-O-dimethyladenosine (m⁶A_m), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m⁶A_m we find that m⁶A_m-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m⁶A_m-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m⁶A_m is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m⁶A_m rather than N⁶-methyladenosine (m⁶A), and reduces the stability of m⁶A_m mRNAs. Together, these findings show that the methylation status of m⁶A_m in the 5' cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability.