Single-base mapping of m6A by an antibody-independent method

Abstract

N6-methyladenosine (m6A) is one of the most abundant mRNA modifications in eukaryotes, involved in various pivotal processes of RNA metabolism. The most popular high-throughput m6A identification method depends on the anti-m6A antibody but suffers from poor reproducibility and limited resolution. Exact location information is of great value for understanding the dynamics, machinery and functions of m6A. Here we developed a precise and high-throughput antibody-independent m6A identification method based on the m6A-sensitive RNA endoribonuclease (m6A-sensitive RNA-Endoribonuclease-Facilitated sequencing or m6A-REF-seq). Whole-transcriptomic, single-base m6A maps generated by m6A-REF-seq quantitatively displayed an explicit distribution pattern with enrichment on stop codons. Independent methods were used to validate the methylation status and abundance of individual m6A sites, confirming the high reliability and accuracy of m6A-REF-seq. We applied this method on five tissues from human, mouse and rat, showing that m6A sites were conserved with single nucleotide specificity and tend to cluster among species.