Acute depletion of METTL3 identifies a role for N6-methyladenosine in alternative intron/exon inclusion in the nascent transcriptome

Abstract

RNA N6-methyladenosine (m6A) modification plays important roles in multiple aspects of RNA regulation. m6A is installed co-transcriptionally by the METTL3/14 complex, but its direct roles in RNA processing remain unclear. Here we investigate the presence of m6A in nascent RNA of mouse embryonic stem cells (mESCs). We find that around 10% m6A peaks are in introns, often close to 5-splice sites. RNA m6A peaks significantly overlap with RBM15 RNA binding sites and the histone modification H3K36me3. Interestingly, acute dTAG depletion of METTL3 reveals that inclusion of m6A-bearing alternative introns/exons in the nascent transcriptome is disrupted. For terminal or variable-length exons, m6A peaks are generally located upstream of a repressed 5-splice site, and downstream of an enhanced 5-splice site. Intriguingly, genes with the most immediate effects on splicing include several components of the m6A pathway, suggesting an autoregulatory function. Our findings demonstrate a direct crosstalk between m6A machinery and the regulation of RNA processing.