m6A level and isoform characterization sequencing (m6A-LAIC-seq) reveals the census and complexity of the m6A epitranscriptome

摘要

N⁶-Methyladenosine (m⁶A) is a widespread, reversible chemical modification of RNA molecules, implicated in many aspects of RNA metabolism. Little quantitative information exists as to either how many transcript copies of particular genes are m⁶A modified (‘m⁶A levels’) or the relationship of m⁶A modification(s) to alternative RNA isoforms. To deconvolute the m⁶A epitranscriptome, we developed m⁶A-level and isoform-characterization sequencing (m⁶A-LAIC-seq). We found that cells exhibit a broad range of nonstoichiometric m⁶A levels with cell-type specificity. At the level of isoform characterization, we discovered widespread differences in the use of tandem alternative polyadenylation (APA) sites by methylated and nonmethylated transcript isoforms of individual genes. Strikingly, there is a strong bias for methylated transcripts to be coupled with proximal APA sites, resulting in shortened 3′ untranslated regions, while nonmethylated transcript isoforms tend to use distal APA sites. m⁶A-LAIC-seq yields a new perspective on transcriptome complexity and links APA usage to m⁶A modifications.