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bioRxiv · 10.1101/2021.05.11.443654

O-GlcNAcylation reduces phase separation and aggregation of the EWS N-terminal low complexity region

Nosella, M. L.·Tereshchenko, M.·Pritisanac, I.·Chong, A.·Toretsky, J. A.·Lee, H. O.·Forman-Kay, J. D.
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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 {beta}-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 and more prone to aggregation based on 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. Insert Table of Contents artwork here O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/443654v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@a09fb6org.highwire.dtl.DTLVardef@1556f47org.highwire.dtl.DTLVardef@1fcd76borg.highwire.dtl.DTLVardef@a2dbda_HPS_FORMAT_FIGEXP M_FIG C_FIG For Table of Contents only.

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Nosella, M. L., Tereshchenko, M., Pritisanac, I., Chong, A., Toretsky, J. A., Lee, H. O., Forman-Kay, J. D.. 2021-05-11. O-GlcNAcylation reduces phase separation and aggregation of the EWS N-terminal low complexity region. https://doi.org/10.1101/2021.05.11.443654

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