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

Abstract

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. The heterogeneity of isoform level m6A RNA methylation in single cells is unclear. The authors characterize m6A at both single-cell and isoform level through ONT long-read sequencing on single-cell cDNA library with APOBEC1-YTH induced C-to-U mutations. They find the role of m6A on surveillance of misprocessed RNAs through CDS-m6A decay mechanism.