Single-molecule m6 A detection empowered by endogenous labeling unveils complexitiesacross RNA isoforms

N⁶-methyadenosine (m⁶A) is a prevalent and dynamic modification on mRNAs and diverse types of noncoding RNAs. 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 by using GeneMind SURFSeq 5000 sequening platform. 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.

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