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

• Published in: Journal of the American Chemical Society Vol. 143; no. 30; pp. 11520 - 11534
• Main Authors: Nosella, Michael L, Tereshchenko, Maria, Pritišanac, Iva, Chong, P. Andrew, Toretsky, Jeffrey A, Lee, Hyun O, Forman-Kay, Julie D
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 04.08.2021
• Subjects: Acetylglucosamine - chemistry; Acetylglucosamine - metabolism; Biomolecular Condensates; HeLa Cells; Humans; Protein Domains; Protein Processing, Post-Translational; RNA-Binding Protein EWS - chemistry; RNA-Binding Protein EWS - metabolism; Tumor Cells, Cultured
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.1c04194

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 (LCRN) 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 LCRN condensates and causes the droplets to exhibit more liquid-like relaxation following fusion. Following extended incubation times, EWS LCRN+RBD condensates exhibit diminished FRAP, indicating a loss of fluidity, while condensates containing the O-GlcNAcylated LCRN 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.