Single-molecule m6A detection empowered by endogenous labeling unveils complexities across RNA isoforms

The landscape of m6A on different RNA isoforms is still incompletely understood. Here, in HEK293T cells, we endogenously label the methylated m6A sites on single ONT DRS reads by APOBEC1-YTH induced C-to-U mutations 10~100 nt away, obtaining 1,020,237 5-mer single-read m6A signals. We then trained m6Aiso, a deep residual neural network model that accurately identifies and quantifies m6A at single-read resolution. Analyzing m6Aiso-determined m6A on single reads and isoforms uncovers distance-dependent linkages of m6A sites along single molecules. It also uncovers specific methylation of identical m6A sites on intron-retained isoforms, partly due to their differential distances to exon junctions and isoform-specific binding of TARBP2. Moreover, we find transcription factor SMAD3 promotes m6A deposition on its transcribed RNA isoforms during epithelial-mesenchymal transition, resulting in isoform-specific regulation of m6A on isoforms with alternative promoters. Our study underscores effectiveness of m6Aiso in elucidating the intricate dynamics and complexities of m6A across RNA isoforms.