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

Abstract

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. <i>O</i>-linked β-D-<i>N</i>-acetylglucosaminylation (<i>O</i>-GlcNAcylation) is an abundant form of intracellular glycosylation whose roles in regulating biomolecular condensate assembly and dynamics have not been delineated. Using an <i>in vitro</i> approach, we found that <i>O</i>-GlcNAcylation reduces the phase separation propensity of the EWS <i>N</i>-terminal low complexity region (LCR_N) under different conditions, including in the presence of the arginine- and glycine-rich RNA-binding domains (RBD). <i>O</i>-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 <i>O</i>-GlcNAcylated LCR_N do not. In HeLa cells, EWS is less <i>O</i>-GlcNAcylated following <i>OGT</i> knockdown, which correlates with its increased accumulation in a filter retardation assay. Relative to the human proteome, <i>O</i>-GlcNAcylated proteins are enriched with regions that are predicted to phase separate, suggesting a general role of <i>O</i>-GlcNAcylation in regulation of biomolecular condensates.