Citation

  • Authors: Nosella ML. et al.
  • Year: 2021
  • Journal: J Am Chem Soc 143 11520-11534
  • Applications: in vitro / siRNA / INTERFERin
  • Cell type: HeLa
    Description: Human cervix epitheloid carcinoma cells

Method

To downregulate expression of OGT, cells were reverse-transfected with 20 nM ON-TARGETplus Human OGT siRNA for 48 h using INTERFERin siRNA Transfection Reagent (Polyplus). Knockdown efficiency was verified by immunoblotting with an anti-OGT antibody, while global O-GlcNAc levels were assessed by immunoblotting with an anti-O-GlcNAc antibody.

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.

Pubmed