Deciphering the ‘m6A code’ via quantitative profiling of m6A at single-nucleotide resolution [III]. Deciphering the ‘m6A code’ via quantitative profiling of m6A at single-nucleotide resolution [III]

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: 17 samples were analyzed: FTO over expression and control in hESC with triplicates; AlkBH5 over-expression, FTO over-expression and control in HEK293T with triplicates; Cytosolic fraction of HEK293T with duplicates

N6-甲基腺嘌呤（N6-methyladenosine, m6A）是信使RNA（messenger RNA, mRNA）上分布最为广泛的转录后修饰，其在发育进程、生理调控及疾病发生中均发挥关键作用。该领域长期面临两大核心难题：一是无法精准定量m6A的化学计量比，二是缺乏不依赖抗体的m6A检测研究方法。本研究基于核糖核酸酶（RNAse）对甲基化位点的差异性切割特性，开发了MASTER-seq技术，可实现单核苷酸分辨率下m6A的系统性定量谱分析。该技术不仅可验证并从头发现m6A修饰位点，还能校准基于抗体的m6A检测方法的性能，并可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。研究发现，m6A的化学计量比可通过一套简单且可预测的顺式编码规则固有决定，该规则可解释约50%的甲基化水平变异，并能精准预测进化过程中m6A的丢失或获得事件。MASTER-seq技术将为不同细胞类型与疾病状态下的m6A调控机制定量研究提供有力工具。
整体实验设计：共分析17个样本：人胚胎干细胞（human embryonic stem cell, hESC）中FTO过表达组及对照组，设置3次生物学重复；HEK293T细胞中AlkB同源物5（AlkB homolog 5, AlkBH5）过表达组、FTO过表达组及对照组，设置3次生物学重复；HEK293T细胞的细胞质组分样本，设置2次生物学重复。