De novo detection of m6A modification in Saccharomyces cerevisiae at single nucleotide resolution

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: 8 samples are analyzed: IP and background for IME4 mutant and WT with 2 biological replicates for each condition

N6-甲基腺嘌呤（N6-methyladenosine，m⁶A）是信使RNA（messenger RNA，mRNA）上含量最为丰富的转录后修饰，其在生物体发育、生理活动及疾病进程中发挥关键调控作用。该领域长期面临的核心挑战在于无法精准定量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。本研究依托核糖核酸酶（RNAse）在甲基化位点的差异化切割特性，开发出MASTER-seq技术，可在单核苷酸分辨率下对m⁶A进行系统性定量谱型分析。借助该技术，研究人员可实现m⁶A位点的验证与从头发现、抗体依赖检测方法的性能校准，以及对酵母配子发生与哺乳动物细胞分化过程中m⁶A动态变化的定量追踪。本研究发现，m⁶A的化学计量比可通过一套简单且可预测的顺式作用编码规则被“硬编码”。该编码规则可解释约50%的甲基化水平差异，并能准确预测进化过程中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强有力的工具。实验整体设计：共分析8个样本，涵盖IME4突变体与野生型（Wild Type，WT）的免疫沉淀（Immunoprecipitation，IP）样本及背景对照，每组条件均设置2次生物学重复。