Molecular grammar of RNA m ⁶ A modification in ribonucleoprotein granules

Diverse RNA chemical modifications collectively regulate nearly all aspects of RNA metabolism. Among these, N ⁶ -methyladenosine (m ⁶ A) is the most prevalent internal modification in eukaryotic messenger RNAs. Recent studies have implicated m ⁶ A 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 m ⁶ A modification ratio. Using this approach, we show that physiological m ⁶ A 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 m ⁶ A modulates condensate dynamics and suggest that RNA modifications can encode regulatory information to tune RNP granule behavior, with potential implications for therapeutic intervention.