Isoform characterization of m(6)A in single cells identifies its role in RNA surveillance.

The distribution of m(6)A across various RNA isoforms and its heterogeneity within single cells are still not well understood. Here, we develop m(6)A-isoSC-seq, which employs both Oxford Nanopore long-read and Illumina short-read sequencing on the same 10x Genomics single-cell cDNA library with APOBEC1-YTH induced C-to-U mutations near m(6)A sites. Through m(6)A-isoSC-seq on a pooled sample of three cell line origins, we unveil a profound degree of m(6)A heterogeneity at both the isoform and single-cell levels. Through comparisons across single cells, we identify widespread specific m(6)A methylation on certain RNA isoforms, usually those misprocessed RNA isoforms. Compared to the coding isoforms of the same genes, the expression of highly methylated misprocessed RNA isoforms is more sensitive to METTL3 depletion. These misprocessed RNAs tend to have excessive m(6)A sites in coding regions, which are targets of CDS-m(6)A decay (CMD). This study offers undocumented insights into the role of m(6)A in RNA surveillance.