Interactions between RNA m6A modification, alternative splicing, and poly(A) tail revealed by MePAIso-seq2

RNA post-transcriptional regulation involves 5'-end capping, 3' poly(A) tailing (including polyadenylation sites, tail length, and non-A residues1, 2), alternative splicing, and chemical modifications including N6-methyladenosine (m6A)3. Studying the interplay of m6A, alternative splicing, alternative polyadenylation sites, poly(A) tail length, and non-A residues in poly(A) tails requires monitoring them simultaneously on one transcript, however strategies to achieve this are lacking. Therefore, we developed a new method, combining m6A-specific methylated RNA immunoprecipitation and the PacBio-based, tail-included, full-length RNA sequencing approach PAIso-seq2, which weve named m6A and poly(A) inclusive RNA isoform sequencing 2 (MePAIso-seq2). Using MePAIso-seq2, we revealed that m6A promotes and inhibits a similar number of alternative splicing events in mouse cell lines, showing that m6A does affect alternative splicing. In contrast, no correlation was detected between m6A and alternative polyadenylation sites choice. Surprisingly, we found that m6A-modified RNAs possess longer poly(A) tails and a lower proportion of poly(A) tails containing non-A residues, especially in mouse embryonic stem cells. Together, we developed a new method to detect full-length m6A-modified RNAs to comprehensively study the relationships between m6A, alternative splicing, and poly(A) tailing, laying a foundation for further exploration of the functional coordination of different RNA post-transcriptional modifications.