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

摘要

Nsup6/sup-methyladenosine (msup6/supA) is one of the most abundant messenger RNA modifications in eukaryotes involved in various pivotal processes of RNA metabolism. The most popular high-throughput msup6/supA identification method depends on the anti-msup6/supA antibody but suffers from poor reproducibility and limited resolution. Exact location information is of great value for understanding the dynamics, machinery, and functions of msup6/supA. Here, we developed a precise and high-throughput antibody-independent msup6/supA identification method based on the msup6/supA-sensitive RNA endoribonuclease recognizing ACA motif (msup6/supA-sensitive RNA-Endoribonuclease–Facilitated sequencing or msup6/supA-REF-seq). Whole-transcriptomic, single-base msup6/supA maps generated by msup6/supA-REF-seq quantitatively displayed an explicit distribution pattern with enrichment near stop codons. We used independent methods to validate methylation status and abundance of individual msup6/supA sites, confirming the high reliability and accuracy of msup6/supA-REF-seq. We applied this method on five tissues from human, mouse, and rat, showing that msup6/supA sites are conserved with single-nucleotide specificity and tend to cluster among species.