Isoform characterization of m⁶A in single cells identifies its role in RNA surveillance

The distribution of m⁶A across various RNA isoforms and its heterogeneity within single cells are still not well understood. Here, we develop m⁶A-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 m⁶A sites. Through m⁶A-isoSC-seq on a pooled sample of three cell line origins, we unveil a profound degree of m⁶A heterogeneity at both the isoform and single-cell levels. Through comparisons across single cells, we identify widespread specific m⁶A 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 m⁶A sites in coding regions, which are targets of CDS-m⁶A decay (CMD). This study offers undocumented insights into the role of m⁶A in RNA surveillance.