Single base mapping of m6A by an antibody-independent method

N6-methyladenosine (m6A) is one of the most abundant mRNA modifications in eukaryotes, related to pivotal RNA metabolism processes. The most popular high-throughput m6A identification method relies on the commercial m6A antibody but suffers from poor reproducibility and limited resolution. Exact location of m6A site is of great vital for understanding the dynamics, functions and machinery of RNA methylation. Here, we developed a precise and high-throughput antibody-independent m6A identification method based on the m6A-sensitive RNA endoribonuclease recognizing ACA motif (m6A-sensitive RNA-Endoribonuclease–Facilitated sequencing or m6A-REF-seq). Whole-transcriptomic single base m6A map generated by m6A-REF-seq displayed a typical distribution pattern with enrichment adjacent to stop codon. Ligase-based and qPCR validation methods were used to confirm the individual m6A sites and quantify the methylation level, reinforcing the high accuracy of m6A-REF-seq. We applied m6A-REF-seq on five tissues from three mammals, showing that m6A sites were conserved and tend to gather together among species. (m6A-REF-seq had been named as Aim-seq.)N6-甲基腺嘌呤（N6-methyladenosine, m6A）是真核生物中最丰富的信使RNA（mRNA）修饰之一，与关键的RNA代谢过程密切相关。当前主流的高通量m6A鉴定方法依赖商业化的m6A抗体，但存在重复性差、分辨率有限的缺陷。精准定位m6A位点对于理解RNA甲基化的动态变化、生物学功能及作用机制至关重要。本研究开发了一种基于识别ACA基序的m6A敏感RNA核糖核酸内切酶的无抗体精准高通量m6A鉴定方法，即m6A敏感RNA核糖核酸内切酶辅助测序（m6A-sensitive RNA-Endoribonuclease–Facilitated sequencing，简称m6A-REF-seq，曾命名为Aim-seq）。通过m6A-REF-seq构建的全转录组单碱基分辨率m6A图谱呈现出典型的分布特征，即在终止密码子附近富集。研究采用基于连接酶的验证方法与qPCR验证方法，对单个m6A位点进行验证并量化甲基化水平，证实了m6A-REF-seq的高准确性。我们将m6A-REF-seq应用于三种哺乳动物的五种组织，结果显示m6A位点在物种间具有保守性，且倾向于聚集分布。