m⁶A mRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover

Understanding the biologic role of N⁶-methyladenosine (m⁶A) 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 m⁶A in exons but very rarely in introns. The m⁶A methylation is essentially completed upon the release of mRNA into the nucleoplasm. Furthermore, the content and location of each m⁶A 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 m⁶As in CA-RNA are within 50 nucleotides of 5' or 3' splice sites, and the vast majority of exons harboring m⁶A in wild-type mouse stem cells is spliced the same in cells lacking the major m⁶A methyltransferase Mettl3. Both HeLa and mouse embryonic stem cell mRNAs harboring m⁶As 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, m⁶A is added to exons before or soon after exon definition in nascent pre-mRNA, and while m⁶A is not required for most splicing, its addition in the nascent transcript is a determinant of cytoplasmic mRNA stability.