High-stoichiometry m⁶A sites are evolutionarily conserved

Area of Science:

• Molecular Biology
• Evolutionary Biology
• Genomics

Background:

• N6-methyladenosine (m6A) is the most abundant internal mRNA modification in eukaryotes.
• The functional significance and evolutionary conservation of m6A sites remain debated, with conflicting previous findings.

Purpose of the Study:

• To definitively determine if m6A sites are under evolutionary selection.
• To reconcile inconsistent results from previous evolutionary analyses of m6A sites.

Main Methods:

• Development of novel motif-level conservation metrics incorporating m6A biogenesis.
• Analysis of ~500,000 m6A sites using quantitative, single-nucleotide resolution measurements across 447 mammalian species.
• Controlling for proximity to exon-junctions to isolate selection pressures on m6A sites.

Main Results:

• A clear, dose-dependent relationship between m6A stoichiometry and evolutionary conservation was observed in both coding sequences (CDS) and untranslated regions (UTRs).
• Highly methylated m6A sites (>60%) displayed significantly greater conservation than lowly methylated sites.
• The observed conservation provides definitive evidence for purifying selection acting on m6A sites.

Conclusions:

• Highly m6A-modified sites are functionally important for gene regulation, challenging the notion of neutral byproduct.
• Established a methodological framework for evolutionary analysis of RNA modifications using quantitative data and phylogenetic sampling.