Determinants of the m6A Epitranscriptome

N6–methyladenosine (m6A) is the most abundant mRNA modification and is crucial for gene expression regulation in numerous biological processes. In mammals, m6A methylation is specifically deposited on a subset of mRNAs and is strongly enriched within long internal exons and near stop codons. This specificity is central to m6A-mediated gene expression regulation, as m6A reader proteins bind m6A methylated regions of transcripts and exert downstream effects on gene expression. However, the basis of this specificity is poorly understood. Here, we develop a Massively Parallel assay for m6A (MPm6A) to systematically elucidate determinants of mRNA m6A specificity. Unexpectedly, we discover that m6A specificity is globally regulated by m6A "suppressors" that prevent m6A deposition in unmethylated transcriptome regions. We find that spliceosomes selectively suppress m6A methylation in an exon length-dependent manner by depositing Exon Junction Complexes (EJC) that protect exon junction-proximal RNA from methylation. EJC suppression of m6A underlies multiple global characteristics of m6A specificity. EJC depletion causes aberrant methylation of thousands of mRNAs, resulting in widespread m6A-mediated gene expression dysregulation. Altogether, we demonstrate that m6A suppression by EJCs is a major mechanistic determinant of m6A epitranscriptome specificity. Furthermore, we find that EJC-suppressed methylation sites co-localize with EJC-suppressed splice sites, suggesting that EJCs broadly suppress local mRNA accessibility to regulatory machineries by packaging proximal RNA.