N6-Methyladenosine mediates alternative intron/exon inclusion in the nascent transcriptome [4sU-seq]

N6-methyladenosines (m6A) are stoichiometrically deposited on exons of nearly one third of RNA Pol II transcriptome, mainly catalysed by METTL3/14 complex. However, neither the intronic methylation pattern and its functional relevance nor the immediate response upon m6A loss have been fully understood. Here we applied MeRIP-seq on mESC nascent transcriptome and thus revealed that approximately 6-10% of m6A peaks occurred at intronic regions, preferentially the conserved and alternative exon/intron part of longer introns, proximately to 5’-splice sites. These intronic m6A deposition correlates with both Rbm15 binding and H3K36me3. Moreover, coupled 4sU-seq with METTL3 dTAG system in mESC, we found that m6A mediates alternative exon/intron inclusive in nascent transcriptome. Intriguingly, m6A self-regulation including writers and readers are early response and partially contributed by alternative splicing changes. Collectively, our study presents a unified model that m6A mediates alternative splicing, and dTAG METTL3 opens an avenue to interrogate the direct response of functional m6A disruption. dTAG (FKBP12F36V) was inserted into Mettl3 C-terminus. Mettl3_FKBP12F36 is very sensitive to dTAG-13 treatment. With this system, we investigated the immediate response of functional m6A loss on nascent transcriptome by 4sU-seq. Cells (2 clones and 3 independent replicates in total) are either treated with dTAG-13 or not for 3 hours, then 4sU was incorporated into the medium for 30min. The 4sU-contaning RNAs were isolated and subjected to sequencing.