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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.