Deciphering the m6a code via antibody-independent quantitative profiling

N6-methyladenosine (m⁶A) is the most abundant modification on mRNA and is involved in critical roles in development, physiology, and disease. The ability to map m⁶A using immunoprecipitation-based approaches has played a critical role in dissecting m⁶A functions and mechanisms of action. Yet, these technologies are of limited specificity, unknown sensitivity, and unable to quantify m6A stoichiometry. These constraints have limited our ability to unravel the factors determining where m⁶A will be deposited, to which levels (the ‘m⁶A code’), and to quantitatively profile m⁶A dynamics across biological systems. Here, we used the RNase MazF, which cleaves specifically at unmethylated RNA sites, to develop MASTER-seq for systematic quantitative profiling of m⁶A sites at 16-25% of all m⁶A sites at single-nucleotide resolution. We established MASTER-seq for orthogonal validation and de novo detection of m⁶A sites, and for tracking of m⁶A dynamics in yeast gametogenesis and in early mammalian differentiation. We discovered that antibody-based approaches severely underestimate the number of m⁶A sites and that both the presence of m⁶A and its stoichiometry are ‘hard-coded’ via a simple and predictable code in cis. This code accounts for ~50% of the variability in methylation levels across sites, allows excellent de novo prediction of methylation sites, and predicts methylation acquisition and loss across evolutionarily distant species. We anticipate that MASTER-seq will pave the path towards a more rigorous quantitative investigation of m⁶A biogenesis and regulation in a wide variety of systems, including diverse cell types, stimuli, subcellular components, and disease states.