Determinant of m6A regional preference by transcriptional dynamics

N6-Methyladenosine (m6A) is the most abundant chemical modification occurring on eukaryotic mRNAs, and has been reported to be involved in almost all stages of mRNA metabolism. The distribution of m6A sites is notably asymmetric along mRNAs, with a strong preference toward the 3' terminus of the transcript. How m6A regional preference is shaped remains incompletely understood. In this study, by performing m6A-seq on chromatin-associated RNAs, we found that m6A regional preference arises during transcription. Nucleosome occupancy is remarkedly increased in the region downstream of m6A sites, suggesting an intricate interplay between m6A methylation and nucleosome-mediated transcriptional dynamics. Notably, we found a remarkable slowdown of Pol-II movement around m6A sites. In addition, inhibiting Pol-II movement increases nearby m6A methylation levels. By analyzing massively parallel assays for m6A, we found that RNA secondary structures inhibit m6A methylation. Remarkably, the m6A sites associated with Pol-II pausing tend to be embedded within RNA secondary structures. These results suggest that Pol-II pausing could affect the accessibility of m6A motifs to the methyltransferase complex and subsequent m6A methylation by mediating RNA secondary structure. Overall, our study reveals a crucial role of transcriptional dynamics in the formation of m6A regional preference.