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