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

HeLa cell chromatin associated nascent pre-mRNA (CA-RNA) contains many unspliced introns and m6A in exons, but very rarely in introns. m6A methylation of exons in CA-RNA frequently occur before splicing of exons and is essentially completed upon the release of mRNA into the nucleoplasm. m6A modifications are virtually the same in the newly synthesized CA-RNA, nucleoplasmic mRNA in transit and in the steady state cytoplasmic mRNA. This result suggests quantitatively little cytoplasmic methylation or demethylation. Only ~10% of m6As in CA-RNA are within 50 nucleotides of 5'' or 3'' splice sites arguing against a common splicing function of methylation. Both HeLa and mouse embryonic stem cell mRNAs showed a distinct increase of m6As, some in clusters, in short-lived mRNAs. Thus there is an unanticipated observation that m6A is added to exons presumably coincident with or soon after exon definition in nascent pre-mRNA, linking nuclear methylation to cytoplasmic mRNA stability. Overall design: Precise mapping m6A sites in human HeLa cells (CA-RNA, nucleoplasmic RNA and cytoplasmic RNAs) and mouse embryonic stem (ES) cells by m6A-CLIP/IP (briefly m6A-CLIP); mRNA half-life quantification by Actinomycin-D treatment and followed with RNA-Seq at different time points with biological replicates in mouse ES cells; RNAseq of HeLa CA-RNA (Ribo 0), nucleoplasmic RNA (Ribo 0), cytoplasmic RNA (Ribo 0) and cytoplasmic RNA (polyA+)