Deciphering the 'm6A code' via quantitative profiling of m6A at single-nucleotide resolution [II]

N6-methyladenosine (m6A) is the most abundant modification on mRNA, and is implicated in critical roles in development, physiology and disease. A major challenge in the field has been the inability to quantify m6A stoichiometry and the lack of antibody-independent methodologies for interrogating m6A. Here, we develop MASTER-seq for systematic quantitative profiling of m6A at single nucleotide resolution, building on differential cleavage by an RNAse at methylated sites. MASTER-seq permitted validation and de novo discovery of m6A sites, calibration of the performance of antibody based approaches, and quantitative tracking of m6A dynamics in yeast gametogenesis and mammalian differentiation. We discover that m6A stoichiometry is 'hard-coded' in cis via a simple and predictable code. This code accounts for ~50% of the variability in methylation levels and allows accurate prediction of m6A loss/acquisition events across evolution. MASTER-seq will allow quantitative investigation of m6A regulation in diverse cell types and disease states. Overall design: 10 samples were analyzed: EBS WT and Metll3 -/- with two replicates each and ESC WT and Mettld -/- with three replicates

N6-甲基腺嘌呤（N6-methyladenosine，m6A）是信使RNA（messenger RNA，mRNA）上最为丰富的表观转录修饰类型，其在个体发育、生理过程及疾病发生中发挥关键调控作用。
该领域长期面临的核心挑战之一，是无法定量测定m6A的化学计量比，且缺乏不依赖抗体的研究手段以解析m6A修饰。
本研究基于核糖核酸酶（ribonuclease，RNase）在甲基化位点的差异化切割特性，开发出MASTER-seq技术，可实现单核苷酸分辨率下的m6A系统性定量图谱分析。
借助MASTER-seq技术，我们能够完成m6A位点的验证与从头发现，校准基于抗体的检测方法的性能，并定量追踪酵母配子发生与哺乳动物细胞分化过程中的m6A动态变化。
本研究发现，m6A的化学计量比可通过一套简洁且可预测的顺式编码规则被“固化编码”。
这套编码规则可解释约50%的甲基化水平变异，并能精准预测跨演化进程中m6A的丢失或获得事件。
MASTER-seq技术将助力在多种细胞类型及疾病状态下开展m6A调控机制的定量研究。
实验整体设计：共分析10份样本：野生型（wild type，WT）EBS与Metll3基因纯合敲除（Metll3 -/-）样本各设2次生物学重复，野生型（WT）胚胎干细胞（embryonic stem cell，ESC）与Mettld基因纯合敲除（Mettld -/-）样本各设3次生物学重复。