Deciphering the “m⁶A Code” via Antibody-Independent Quantitative Profiling

N6-methyladenosine (m⁶A) is the most abundant modification on mRNA and is implicated in critical roles in development, physiology, and disease. A major limitation has been the inability to quantify m⁶A stoichiometry and the lack of antibody-independent methodologies for interrogating m⁶A. Here, we develop MAZTER-seq for systematic quantitative profiling of m6A at single-nucleotide resolution at 16%–25% of expressed sites, building on differential cleavage by an RNase. MAZTER-seq permits 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, accounting for 33%–46% of the variability in methylation levels and allowing accurate prediction of m⁶A loss and acquisition events across evolution. MAZTER-seq allows quantitative investigation of m⁶A regulation in subcellular fractions, diverse cell types, and disease states.