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

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 do- mains (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.

DNA damage-induced YTHDC1 O-GlcNAcylation promotes homologous recombination by enhancing N6-methyladenosine binding

“…For instance, neuronal SynGAP T1306 O-GlcNAc has been demonstrated to inhibit the formation of the SynGAP-PSD-95 complex, and thus LLPS (33). In addition, O-GlcNAc would attenuate LLPS of the RNA-binding EWS N-terminal disordered region (32). In our work, under basal conditions, we also found that O-GlcNAcylation inhibited YTHDC1 LLPS (Fig.…”

The Nuclear Pore Complex: Birth, Life, and Death of a Cellular Behemoth

“…Indeed, DNAJB6 and DNAJB2 display disaggregation activity towards FG repeats of NUPs in vitro [128] and might thus act as sensors for and keepers of the state of FG repeats in vivo. A glycosylation present on many NUPs across metazoa, O-linked N-acetylglucosamine (O-GlcNAc), may also contribute to functional FG repeat domains and NUP stability, since a reduction of O-GlcNAc modifications was observed to promote proteasome-mediated turn-over of these NUPs [257][258][259]. The large number of intrinsically disordered domains may pose a particular challenge to NPC homeostasis and multiple pathways may contribute to their maintenance.…”