O -Linked- N -Acetylglucosaminylation of the RNA-Binding Protein EWS N-Terminal Low Complexity Region Reduces Phase Separation and Enhances Condensate Dynamics.

Many membraneless organelles are thought to be biomolecular condensates formed by phase separation of proteins and other biopolymers. Post-translational modifications (PTMs) can impact protein phase separation behavior, although for many PTMs this aspect of their function is unknown. O -linked β-D- N -acetylglucosaminylation ( O -GlcNAcylation) is an abundant form of intracellular glycosylation whose roles in regulating biomolecular condensate assembly and dynamics have not been delineated. Using an in vitro approach, we found that O -GlcNAcylation reduces the phase separation propensity of the EWS N -terminal low complexity region (LCR _N ) under different conditions, including in the presence of the arginine- and glycine-rich RNA-binding domains (RBD). O -GlcNAcylation enhances fluorescence recovery after photobleaching (FRAP) within EWS LCR _N condensates and causes the droplets to exhibit more liquid-like relaxation following fusion. Following extended incubation times, EWS LCR _N +RBD condensates exhibit diminished FRAP, indicating a loss of fluidity, while condensates containing the O -GlcNAcylated LCR _N do not. In HeLa cells, EWS is less O -GlcNAcylated following OGT knockdown, which correlates with its increased accumulation in a filter retardation assay. Relative to the human proteome, O -GlcNAcylated proteins are enriched with regions that are predicted to phase separate, suggesting a general role of O -GlcNAcylation in regulation of biomolecular condensates.