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

Garcia-Campos, Miguel Angel, Edelheit, Sarit, Toth, Ursula, Safra, Modi, Shachar, Ran, Viukov, Sergey, Winkler, Roni, Nir, Ronit, Lasman, Lior, Brandis, Alexander, Hanna, Jacob H., Rossmanith, Walter, Schwartz, Schraga
Cell 2019 v.178 no.3 pp. 731-747.e16
evolution, gametogenesis, mammals, messenger RNA, methylation, prediction, ribonucleases, stoichiometry, subcellular fractions, yeasts
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.