mA mRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover.

Understanding the biologic role of -methyladenosine (mA) RNA modifications in mRNA requires an understanding of when and where in the life of a pre-mRNA transcript the modifications are made. We found that HeLa cell chromatin-associated nascent pre-mRNA (CA-RNA) contains many unspliced introns and mA in exons but very rarely in introns. The mA methylation is essentially completed upon the release of mRNA into the nucleoplasm. Furthermore, the content and location of each mA modification in steady-state cytoplasmic mRNA are largely indistinguishable from those in the newly synthesized CA-RNA or nucleoplasmic mRNA. This result suggests that quantitatively little methylation or demethylation occurs in cytoplasmic mRNA. In addition, only ∼10% of mAs in CA-RNA are within 50 nucleotides of 5' or 3' splice sites, and the vast majority of exons harboring mA in wild-type mouse stem cells is spliced the same in cells lacking the major mA methyltransferase Mettl3. Both HeLa and mouse embryonic stem cell mRNAs harboring mAs have shorter half-lives, and thousands of these mRNAs have increased half-lives (twofold or more) in Mettl3 knockout cells compared with wild type. In summary, mA is added to exons before or soon after exon definition in nascent pre-mRNA, and while mA is not required for most splicing, its addition in the nascent transcript is a determinant of cytoplasmic mRNA stability.