Phosphorylation tunes electrostatically driven protein-RNA interactions

Phosphorylation of intrinsically disordered proteins (IDPs) regulates many biomolecular interactions, yet its quantitative effects on binding of highly charged IDPs to nucleic acids remain unclear. Here, we show that multi-site phosphorylation of the disordered arginine/serine-rich (RS) domain of the splicing factor SRSF1 functions as an electrostatic rheostat controlling RNA binding. Using enzymatic phosphorylation, phosphomimetic variants, chemically synthesized phosphopeptides, and single-molecule Förster resonance energy transfer measurements, we identify the RS domain as a strong driver of protein-RNA association. Progressive phosphorylation gradually weakens this interaction, and extensive phosphorylation abolishes detectable RNA binding. Notably, the binding free energy varies linearly with RS domain net charge, independent of whether the charge arises from phosphorylation or acidic phosphomimetic substitutions. Together, our findings establish a quantitative framework for how phosphorylation tunes interactions between charged IDPs and nucleic acids, and rationalize why two acidic amino acid residues are needed to mimic the effect of a single phosphorylation event.