High-stoichiometry m ⁶ A sites are evolutionarily conserved

<i>N</i> ⁶-methyladenosine (m⁶A) is the most prevalent internal mRNA modification in eukaryotes, yet whether m⁶A sites are functionally important or represent neutral byproducts remains unclear. Previous evolutionary analyses failed to detect consistent conservation signatures at m⁶A sites, and report conflicting patterns of conservation across genic regions, such as the coding sequence (CDS) and untranslated regions (UTRs). To reconcile these inconsistencies and definitively determine whether m⁶A sites are under selection, we developed novel motif-level conservation metrics that incorporate knowledge of m⁶A biogenesis to distinguish m⁶A-specific selection from other confounding sources. We analyzed ∼500,000 candidate sites with quantitative, single-nucleotide resolution m⁶A measurements across a phylogeny spanning 447 mammalian species. After controlling for proximity to exon-junctions, we observed a clear, dose-dependent relationship between m⁶A stoichiometry and evolutionary conservation in both CDS and UTRs. Highly methylated sites (>60%) exhibited significantly increased conservation compared to lowly methylated sites-with an effect size approximately one-third of the typical CDS-UTR difference-providing definitive evidence of purifying selection and supporting a model where highly modified sites contribute functionally to gene regulation. We established a methodological framework for evolutionary analysis of RNA modifications, highlighting the necessity of quantitative measurements, comprehensive phylogenetic sampling, and careful consideration of modification biogenesis.