Targeted stress granule regulation by engineering a non-catalytic O-GlcNAc transferase

Stress granules (SGs) are dynamic ribonucleoprotein condensates whose assembly is driven by multivalent interactions and liquid-liquid phase separation (LLPS) of their component proteins and mRNAs. Related to multiple diseases, SGs are an ideal prototype to study cellular condensates. Understanding their regulatory factors and developing modulation methods are critical to therapeutic development. O-GlcNAc transferase (OGT) has been implicated in SG regulation, while its function beyond O-GlcNAcylation remains unknown. Here, we identified OGT to suppress the LLPS of G3BP1, a key SG protein, and thereby SG assembly. Unexpectedly, this suppression is independent of the OGT enzymatic activity. Also, we repurposed OGT into an SG modulator by generating a fusion protein consisting of its N-catalytic domain and poorly understood intervening domain (NI), coupled with induced proximity modules, such as a nanobody. We demonstrate that this inhibitory effect is achieved via targeted protein immobilization, which rigidifies G3BP1 after prolonged stress. This modular, genetically encoded tool recognizes the domain organization of G3BP1, thus is generalizable to another four proteins featuring similar architecture, suppressing condensate formation with mobility reduction. We also applied this strategy for SG perturbations to reveal SG functions on cellular activities. Our work provides a novel strategy for general SG regulation by interfering material properties of critical SG proteins and offers insights into the cryptic non-catalytic function of OGT.