O-Linked-N-Acetylglucosaminylation of the RNA-Binding Protein EWS N-Terminal Low Complexity Region Reduces Phase Separation and Enhances Condensate Dynamics
- 25 July 2021
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of the American Chemical Society
- Vol. 143 (30), 11520-11534
- https://doi.org/10.1021/jacs.1c04194
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. 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.Funding Information
- Canadian Institutes of Health Research (FDN-148375)
- University of Toronto
- National Cancer Institute (P30 CA051008-16, R01CA233619-01A1)
- Canada Research Chairs
- Children's Cancer Foundation
- Natural Sciences and Engineering Research Council of Canada
- Canadian Cancer Society
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