Isoform characterization of m6A in single cells identifies its role in RNA surveillance [eDART-Seq]

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 mis-processed 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 mis-processed 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 on RNA surveillance. Overall design: The pcDNA3.1-YTH-APOBEC-T2A-EGFP construct was transfected into HEK293T cells, HepG2 cells, and HeLa cells, respectively, using Lipofiter 3.0 (Hanbio) according to the manufacturer's instructions. The medium was refreshed 6 hours after transfection, and the EGFP-positive cells of the three cell lines were isolated, respectively, through cytometry 3 day after transfection. Then, the total RNA of pcDNA3.1-YTH-APOBEC-T2A-EGFP transfected HEK293T, HeLa and HepG2 was harvested using Trizol (Invitrogen) followed by treatment with RNase-Free DNase (NEB) to remove possible DNA contamination, respectively. RNA-seq libraries were prepared with Dynabeads mRNA Purification Kit (Ambion) and TruSeq Stranded mRNA Library Prep Kit (Illumina). Sequencing was performed with Illumina HiSeq 2000 to generate about 55 million strand-specific 150 bp paired-end reads for each cell line.