Molecular grammar of RNA m6A modification in ribonucleoprotein granules

Abstract

Diverse RNA chemical modifications collectively regulate nearly all aspects of RNA metabolism. Among these, N6-methyladenosine (m6A) is the most prevalent internal modification in eukaryotic messenger RNAs. Recent studies have implicated m6A in the formation and regulation of ribonucleoprotein (RNP) granules--biomolecular condensates that organize RNA metabolism in space and time. However, the underlying biophysical mechanisms remain to be elucidated. Here, building on SMART, a single-molecule platform for quantitatively measuring RNP granule assembly across nanometer-to-mesoscale regimes, we develop SMART-epi to enable precise control of the m6A modification ratio. Using this approach, we show that physiological m6A levels inhibit YTHDF2 RNP granule assembly while directing specific RNA-RNA interaction architectures. Moreover, YTHDF1/2/3 exhibit distinct assembly kinetics, suggesting functional divergence. Together, these findings reveal a molecular grammar by which m6A modulates condensate dynamics and suggest that RNA modifications can encode regulatory information to tune RNP granule behavior, with potential implications for therapeutic intervention.