Reversible methylation of m6Am 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 (m6Am), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m6Am we find that m6Am-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m6Am-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m6Am is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m6Am rather than N6-methyladenosine (m6A), and reduces the stability of m6Am mRNAs. Together, these findings show that the methylation status of m6Am in the 5' cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability. Overall design: RNA-Seq was performed on HEK293T cells transfected with scrambled siRNA, or siRNA targeting FTO or ALKBH5, wild-type HEK293T cells or HEK293T cells with CRISPR-inactivated DCP2, HEK293T cells transfected with FLAG-vector or NES-FTO-vector (both untreated and 6h actinomycin D treated) and wildtype and Fto knockout mouse liver. Furthermore, miCLIP was performed on HEK293T cells as previously described (see GSE63753). For RNA-Seq, 2-3 biological replicates were used for each experiment and condition. For miCLIP, 6 biological replicates were used and combined into a single fastq file.