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

The landscape of N⁶-methyadenosine (m⁶A) on different RNA isoforms is still incompletely understood. Here, in HEK293T cells, we endogenously label the methylated m⁶A sites on single Oxford Nanopore Technology (ONT) direct RNA sequencing (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 that 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 the effectiveness of m6Aiso in elucidating the intricate dynamics and complexities of m⁶A across RNA isoforms.