Acute depletion of METTL3 implicates N ⁶-methyladenosine in alternative intron/exon inclusion in the nascent...

RNA N ⁶-methyladenosine (m⁶A) modification plays important roles in multiple aspects of RNA regulation. m⁶A is installed cotranscriptionally 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. We find that around 10% of m⁶A peaks are located in alternative introns/exons, often close to 5' splice sites. m⁶A peaks significantly overlap with RBM15 RNA binding sites and the histone modification H3K36me3. Acute depletion of METTL3 disrupts inclusion of alternative introns/exons in the nascent transcriptome, particularly at 5' splice sites that are proximal to m⁶A peaks. For terminal or variable-length exons, m⁶A peaks are generally located on or immediately downstream from a 5' splice site that is suppressed in the presence of m⁶A and upstream of a 5' splice site that is promoted in the presence of m⁶A. Genes with the most immediate effects on splicing include several components of the m⁶A pathway, suggesting an autoregulatory function. Collectively, our findings demonstrate crosstalk between the m⁶A machinery and the regulation of RNA splicing.