Isoform characterization of mA in single cells identifies its role in RNA surveillance.

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