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

Domain: biology
Published: 2026-01-07
Editorial outcome: major_revision

Abstract
Stress granules (SGs) are disease-relevant dynamic ribonucleoprotein condensates formed by liquid-liquid phase separation (LLPS) of proteins and mRNAs. Understanding their regulators and developing interventions are critical for therapeutic development. O-GlcNAc transferase (OGT) has been implicated in SG regulation, but functions beyond O-GlcNAcylation remain unclear. Here we uncover that, upon induced proximity, OGT suppresses LLPS of the SG marker G3BP1 and thereby SG assembly, independent of its catalytic activity. We repurpose OGT into an SG modulator by fusing its N-catalytic and intervening domains (NI) to induced-proximity modules. This inhibitory effect arises from targeted protein immobilization that rigidifies G3BP1 under prolonged stress. This tool recognizes G3BP1’s domain organization, thus generalizes to four additional proteins featuring similar architectures, suppressing condensate formation with mobility reduction. This modular, genetically encoded strategy enables SG regulation and functional dissection by interfering material properties of critical SG proteins and illuminates the cryptic non-catalytic function of OGT.

Introduction
Stress granules (SGs) are among the most extensively studied membrane‑less cytosolic ribonucleoprotein (RNP) assemblies, driven by the collective interactions of a core network of proteins and RNAs1–3. When cells face stressors such as oxidative damage or heat, translation is inhibited and polysomes disassemble, leading to an accumulation of uncoated mRNA1,4–6. These mRNAs coalesce with RNA‑binding proteins into condensates, namely SGs, via liquid–liquid phase separation (LLPS)6,7. SGs often contain proteins implicated in pathological aggregation, such as FUS, TDP‑43, and other heterogeneous nuclear RNPs (hnRNPs)5,8,9. Growing genetic evidence links aberrant SG dynamics to the pathogenesis of neurodegenerative diseases5,8–11. SGs also contribute to cytoprotective responses during viral infection12–14 and chemo‑/radiotherapy15. As SGs emerge as promising therapeutic targets, understanding the regulatory principles that control their dynamics is essential for developing SG‑specific interventions.

During SG assembly, G3BP1 and its paralog G3BP2 function as molecular switches for LLPS, forming multivalent interactions with various binding partners1,16. Beyond perturbing upstream signaling that triggers SG formation17,18, directly tuning the LLPS properties of core SG components is critical to shaping granule composition and dynamics19. A hierarchy of interactions—oligomerization, RNA binding, and intrinsically disordered regions (IDRs)—governs whether SGs behave like liquids or solids19,20. As exemplified by G3BP1, it contains an N-terminal NTF2-like (NTF2L) domain responsible for dimerization and a C-terminal RNA-binding domain (RBD; comprising an RRM and IDR3), both of which are required for its LLPS and SG formation. Consequently, targeting the NTF2L domain by viral peptide mimetics15 or small molecules21,22, or RNA degradation by the RNase treatment13, directly block SG formation. The long central IDR of G3BP1 also modulates SG dynamics but remains a difficult target for selective manipulation1. Many other condensate‑forming proteins share the modular architecture of G3BP1/216. However, existing regulatory approaches for G3BP1/2—primarily designed to target the NTF2L domain—are not easily transferred to these proteins, leaving few methods to control their dynamics in cells. Moreover, despite the centrality of G3BP1/2, other proteins with positive or negative cooperativity also influence SG assembly, reflecting a dynamic, balanced regulatory network1,16,19.

Post-translational modifications (PTMs) with chemical diversity add another layer of SG regulation20,23,24. Notably, O-linked N-acetylglucosamine (O-GlcNAc)—a stress-responsive monosaccharide modification installed by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA)—has long been implicated in SG biology25–28. Several key factors relating to O-GlcNAc modification, including OGT, were found essential for SG assembly, leading to the discovery that O-GlcNAc modifications of components of the translational machinery promote SG assembly25. However, although robust O-GlcNAc signals were commonly found in SGs, it is difficult to observe a detectable OGT signal within SG foci. This discrepancy led us to explore other possible functions of OGT on SGs beyond maintaining O-GlcNAc homeostasis.

In this study, facilitated by a chemical-induced proximity approach, we unexpectedly uncovered a non-catalytic function of OGT in negatively regulating the material properties of key SG proteins, particularly G3BP1, both in vitro and in cells. We next screened out the non-catalytic intervening domain (Int-D) as the primary domain for the suppression of G3BP1-induced SGs under multiple stressors. Accordingly, we designed a SG-targeted suppression method using a non-catalytic OGT truncation combined with a nanobody or chemical-induced proximity, which we name as STOP. We characterized the working pattern of STOP on SG suppression and revealed that STOP substantially reduced G3BP1’s dynamics by recognizing its domain architecture. STOP is also generalizable to other SG key nodes with similar domain features and enables similar condensation suppression. Our study developed STOP as a versatile tool to reshape the material properties of phase-separated condensates of SG components, allowing for cellular activity perturbation through SG intervention. Our work also provides insightful hints for the non-catalytic function of the previously cryptic Int-D in OGT.

Results
OGT suppresses G3BP1-centric SGs independent of its transferase activity
Given the reported low signal of endogenous OGT within SGs25, we wondered how SGs would be affected if we directly brought OGT into SGs using a chemical-induced proximity approach. Using the CRISPR-Cas9 technique29, we knocked in an FKBPF36V tag at the N-terminus of the OGT genomic locus in HeLa cells, allowing targeting endogenous OGT with an FKBPF36V small molecule ligand AP186730,31 (Supplementary Fig. 1). We overexpressed G3BP1 bearing a C-terminal Halotag (G3BP1-Halo) in this engineered HeLa cell line as the central node and marker protein for SG formation32, which was able to form liquid-like condensates in HeLa cells under oxidative stress induced by 500 µM sodium arsenite for 30 min (Fig. 1a). As expected, endogenous FKBPF36V-OGT was unable to enter G3BP1-Halo–centric SGs under the AP1867 treatment followed by arsenite (Fig. 1b). To achieve chemical-induced proximity between FKBPF36V-OGT and G3BP1-Halo, we synthesized and applied a previously reported small chimeric molecule as a molecular glue—OGTAC-2—containing AP1867 and a haloalkane linking together30 (Fig. 1a and Supplementary Fig. 2a, b). Upon arsenite stress, nearly 60% of cells showed normal SG formation with colocalization of FKBPF36V-OGT and G3BP1-Halo, yet the remaining 40% of cells exhibited substantially reduced SGs, indicating that the direct recruitment of OGT to the SG marker protein, G3BP1, may suppress SG formation in HeLa cells under arsenite stress (Fig. 1c, d). However, due to the overexpression of G3BP1-Halo, endogenous OGT may not be enough to target all G3BP1-Halo proteins in most cells, leading to insufficient SG suppression. Since a previous study defined proteins affecting SG assembly by exhibiting either positive or negative cooperativity1, we speculate that OGT may be one of the factors exhibiting “negative cooperativity” for SG regulation when directly interacting with G3BP1.

Since many identified components in SGs were reported to be O-GlcNAc-modfied substrates, we validated the effects of overexpressed O-GlcNAc cycling enzymes on EGFP-tagged G3BP1 (G3BP1-EGFP) in HeLa cells by monitoring SG assembly under NaAsO2 treatment. Surprisingly, both catalytically active (WT) and inactive (K852A) OGT significantly reduced the number of SG-containing HeLa cells to about 50%, showing a similar SG suppression effect to the chemical-induced G3BP1-OGT proximity. More importantly, this effect is independent of OGT’s transferase activity (Fig. 1e, f). In contrast, co-expression with OGA or its inactive variant (D174N) had little effect on SG formation, with over 95% of cells still forming SGs. Although a potential interaction between G3BP1 and OGT, rather than OGA, was observed (Supplementary Fig. 2c), we failed to detect apparent O-GlcNAc modifications on G3BP1 after either in cells or in vitro glycosylation (Supplementary Fig. 2b–d), further suggesting that the reduction in G3BP1-positive SGs is not directly linked to putative O-GlcNAc modifications on G3BP1.

These in-cell findings prompted us to investigate whether the inhibitory effect of OGT on G3BP1’s LLPS relies on the cellular environment. We performed in vitro droplet formation assays using purified G3BP1-EGFP along with increasing concentrations of purified and Cy5-labeled OGT, where no glycosylation reaction would occur. At a low concentration of 1 µM, OGT was incorporated into G3BP1-EGFP droplets with minimal changes in droplet size compared to G3BP1-EGFP alone (Fig. 1g). However, as the OGT concentration increased to 5 µM and 10 µM, the droplet size dramatically decreased (Fig. 1h). In contrast, EGFP-tagged YTHDF3 (EGFP-YTHDF3), a protein known to undergo phase separation and interact with OGT27, maintained the droplet size and OGT partitioning even at higher OGT concentrations (Supplementary Fig. 2d–f). Taken together, these results imply that OGT specifically inhibits the LLPS of G3BP1 and subsequent SG formation, both in vitro and in cells, through direct interaction independent of its enzymatic activity. This effect becomes more evident as the level of OGT increases. This observation motivates further investigation into the functional domains of OGT and the underlying mechanisms of its action.

Design of a G3BP1-targeted OGT truncation for SG suppression
Given OGT’s ability to suppress G3BP1 LLPS, we next aimed to engineer it into a G3BP1-selective tool for targeted SG regulation with minimized side effects. Previously, we have reported a set of tools for targeted protein O-GlcNAcylation and de-O-GlcNAcylation, utilizing nanobodies for intracellular protein targeting and O-GlcNAc cycling enzymes for modification33–35. In this study, we first employed our previously designed GFP-nanobody-fused OGT variant (nGFP-OGT4) to selectively target G3BP1-EGFP (Fig. 2a). G3BP1-EGFP coexpressed with either catalytically active or inactive nGFP-OGT4 exhibited nearly 40% reduced SG formation after NaAsO2 stress when compared with nGFP-fused truncated OGA (nGFP-OGA(Stalk)) of similar size (Fig. 2b, c). This nanobody-directed G3BP1 targeting reaffirms that the SG suppression effect is irrelevant to the enzymatic activity and size of OGT. Thus, we hypothesized that this effect may depend on the uniqueness of the OGT sequence.

Based on the well-characterized structure of OGT36, we started to identify the key domains responsible for the suppression of G3BP1 phase separation and thereby repurpose OGT into a more compact tool for targeted SG regulation. The HA-tagged nGFP-OGT4 consists of an N-terminal nanobody against EGFP (nGFP), a truncated TPR domain (TPR4), the N-catalytic domain (N-cat), the intervening domain (Int-D), and the C-catalytic domain (C-cat) (Fig. 2d). Accordingly, we generated four variants of nGFP-OGT4 by deleting individual domains and compared their capability to suppress SG formation. Surprisingly, nGFP-OGT4 variants lacking either the TPR or C-cat domain were still able to suppress G3BP1-centric SGs, whereas nGFP-OGT4 variants lacking the N-cat or Int-D domains failed to suppress SG formation, with almost all cells exhibiting regular SG formation, highlighting the importance of the N-cat and Int-D domains (Fig. 2d). To further refine the tool, we fused the individual domains to nGFP for targeting G3BP1-EGFP. Notably, either N-cat or Int-D was sufficient to significantly reduce SG formation. The combination of all three fragments (nGFP, N-cat, and Int-D; termed nGFP-NI hereafter) achieved maximal suppression, reducing SG formation to nearly 90% (Fig. 2e). Given its highest efficacy and compact size, nGFP-NI was used in subsequent experiments as the tool for targeting G3BP1-EGFP in SG regulation. The high affinity between nGFP and EGFP ensured stable interaction between OGT variants and G3BP1 both before and after stress (Supplementary Fig. 3a). However, removal of nGFP from these constructs abolished their capability to suppress SG formation (Supplementary Fig. 3b–e); similarly, nGFP-NI was unable to target endogenous G3BP1 lacking EGFP as well (Supplementary Fig. 3f), both confirming that induced proximity between OGT and G3BP1 is essential for the suppression effect. Consistently, experiments in HeLa cells stably expressing nGFP-OGT4(K852A), its truncation lacking both the N-cat and Int-D domains (nGFP-ΔNI), or the NI fragment alone, confirmed again that both the nGFP moiety and the NI fragment are essential for SG suppression (Supplementary Fig. 4a, b). Furthermore, we evaluated nGFP-NI in two additional cell models: U2OS cells stably expressing G3BP1-EGFP at levels lower than endogenous G3BP1, and U2OS G3BP1/2 double knockout (dKO) cells rescued with G3BP1-EGFP. In both cell lines, nGFP-NI effectively suppressed SG formation under NaAsO2 stress, demonstrating the robustness of the tool (Supplementary Fig. 4c, d).

A recent study from Taylor’s group demonstrated that G3BP1/2 have the highest centrality within the core SG network, which serve as the node for SG assembly under certain conditions, especially arsenite stress1. When applying nGFP-NI for SG suppression, we additionally examined another four known SG markers–eIF4G1, UBAP2L, G3BP2, and USP10–via immunofluorescence to explain the centrality16,37,38. We found that none of these marker proteins formed SGs independently of G3BP1 (Fig. 2f and Supplementary Fig. 5a). These results suggest that targeting G3BP1-EGFP with nGFP-NI prevented the assembly of other key SG marker proteins, reinforcing the central role of G3BP1 in SG formation under arsenite stress. However, it is important to note that G3BP1 may not exhibit the same degree of centrality under other stress conditions. To determine whether nGFP-NI is applicable to SG suppression under different stressors, we tested its effect on G3BP1-EGFP in HeLa cells subjected to osmotic stress (sorbitol) or ER stress (DTT)27. As expected, we observe a significant reduction in G3BP1 granules (Fig. 2g, h). However, unlike the arsenite treatment, other SG markers such as UBAP2L and eIF4G1 formed SGs independent of G3BP1 under sorbitol-induced stress (Supplementary Fig. 5b). These results suggest that nGFP-NI effectively suppresses G3BP1-dependent SGs and provides a strategy for distinguishing the relative importance and centrality of G3BP1 under different stress conditions. Meanwhile, we monitored other membrane less organelles during the application of nGFP-NI to G3BP1-EGFP under arsenite stress. No significant changes were observed in P-bodies or nuclear speckles (Supplementary Fig. 5c–f), underscoring the selectivity of nGFP-NI for its EGFP-tagged target protein and associated condensates.

nGFP-NI suppresses G3BP1 LLPS in an order- and concentration-dependent manner
We next investigated whether nGFP-NI acts directly on G3BP1 or indirectly disrupts SG assembly by modulating broader protein network interactions. We first examined eIF2α phosphorylation (p-eIF2α), a well-characterized trigger for translation inhibition and conventional SG formation in response to stressors18. We observed that both nGFP-NI and nGFP alone, when co-expressed with G3BP1-EGFP, resulted in similar levels of eIF2α phosphorylation in response to arsenite treatment (Supplementary Fig. 6a). Additionally, puromycin detection confirmed that translational arrest occurred as expected under both conditions13,39 (Supplementary Fig. 6b). These data suggest that early steps prior to G3BP1 involvement, such as stress recognition and translational arrest, proceeded normally, which in turn supports the notion that the inhibitory effect of nGFP-NI on SG formation does not stem from interference with the upstream SG signaling pathways, but likely from direct perturbation on G3BP1 itself. To further investigate whether nGFP-NI prevents the initiation of SG assembly or disrupts existing SGs, we employed a chemical-induced proximity approach40 with time and dose controls. We replaced the nGFP–EGFP binding pair with the FKBP–FRB pair, generating G3BP1-FRB and FKBP-NI constructs, respectively. Rapamycin acts as a molecular glue for FKBP and FRB, rapidly forming a ternary complex as its concentration increases without inducing detectable SGs (Supplementary Fig. 7a, b). When Rapamycin was applied at low concentrations prior to arsenite stress, it induced colocalization between G3BP1-FRB and FKBP-NI, while starting to inhibit SG formation once the Rapamycin concentration reached 100 nM or higher (Fig. 3a, b and Supplementary Fig. 7c). Consistent with the aforementioned in vitro droplet formation assay with an increasing amount of purified OGT (Fig. 1g, h), this inhibitory effect on SG is concentration dependent as well. In contrast, when cells were first stressed with arsenite followed by rapamycin, FKBP-NI was successfully recruited into SGs and colocalized with G3BP1-FRB, but SGs persisted (Fig. 3c, d and Supplementary Fig. 7d), indicating that the induced proximity of NI to SGs is unfortunately unable to disassemble existing SGs. These results emphasize that prior interaction between NI and G3BP1 is crucial for the suppression of arsenite-induced SGs. Meanwhile, the use of the Rapamycin-induced FKBP-FRB pair highlights the modularity of nGFP-NI, where the nGFP module can be substituted with other proteins or small molecules for versatile induced proximity.

Furthermore, we conducted in vitro droplet formation assays of G3BP1-EGFP and nGFP-NI triggered by either total RNA or the molecular crowding reagent PEG8000 (Supplementary Fig. 7e, f). After RNA-induced LLPS, G3BP1-EGFP formed spherical droplets, even in the presence of Cy5-labeled nGFP-NI or nGFP (Fig. 3e). However, when G3BP1-EGFP was pre-mixed with nGFP-NI, it became less responsive to RNA-induced LLPS, resulting in significantly smaller droplets (Fig. 3e). Similar results were obtained when 10% PEG8000 was used to trigger G3BP1-EGFP LLPS (Fig. 3f). These results, consistent with our observations in cells, reaffirm that direct interaction between G3BP1 and nGFP-NI prior to stress is essential for inhibiting G3BP1 LLPS and SG formation. Therefore, we sought to examine the interaction between nGFP-NI and G3BP1-EGFP prior to LLPS using cross-linking mass spectrometry (XL-MS). Although a low abundance of lysine residues limited the number of cross-links obtained, we observed multiple cross-links between the NTF2L and RBD domains in the presence of nGFP alone, consistent with previous reports indicating interactions between the N- and C-termini of G3BP11 (Supplementary Fig. 8a). However, in the presence of nGFP-NI, most of these cross-links on G3BP1-EGFP disappeared, while two new cross-links between the NTF2L or RBD domains and nGFP-NI were detected (Supplementary Fig. 8b). These changes suggest that nGFP-NI may sequester G3BP1 and thereby hinder the initiation of G3BP1 LLPS.

nGFP-NI promoted G3BP1 aggregation
Next, to assess whether nGFP-NI prevents or merely delays SG assembly, we monitored SG formation over a two-hour arsenite stress. We observed that at 0.5 h post-arsenite treatment, SGs appeared in control cells, characterized by colocalization of G3BP1-EGFP, the SG marker eIF4G1, and nGFP. In contrast, in cells expressing nGFP-NI, no apparent SGs appeared, and both G3BP1-EGFP and eIF4G1 remained diffusive (Fig. 4a). After 1 h of stress, cytoplasmic puncta appeared in nGFP-NI–expressing cells, sequestering G3BP1-EGFP, eIF4G1, and TDP-43 together (Fig. 4a and Supplementary Fig. 9a). However, while SGs in control cells retained their spherical shape after two hours of stress, the puncta in the nGFP-NI–expressing cells exhibited a more aggregated morphology (Fig. 4a). These observations prompted us to compare the fluidity of these SGs. Fluorescence recovery after photobleaching (FRAP) analyses were conducted following two hours of arsenite treatment and revealed that nGFP-NI significantly slowed the FRAP kinetics of G3BP1-EGFP compared to nGFP, indicating that the G3BP1–NI binary complex exhibited more aggregate-like material properties (Fig. 4b–e and Supplementary Fig. 9b, c). Likewise, FRAP analyses of in vitro G3BP1-EGFP droplets pre-incubated with nGFP or nGFP-NI mirrored these findings, with nGFP-NI dramatically reducing the dynamics of G3BP1-EGFP condensates (Supplementary Fig. 9d–g).

To further corroborate whether the G3BP1-EGFP and nGFP-NI complex formed solid-like aggregates, we treated cells with 1,6-Hexanediol (1,6-HD) following 1-h arsenite stress. In HeLa cells co-expressing nGFP and G3BP1-EGFP, SGs were largely disassembled by 1.5% 1,6-HD, whereas G3BP1-EGFP puncta persisted in the nGFP-NI–expressing cells, suggesting that the puncta formed by the G3BP1–NI binary complex were more solid-like, in contrast to the liquid-like SGs observed in the nGFP group (Fig. 4f). Similar findings were observed on cells when applied a 2-h recovery following 1-h arsenite exposure (Supplementary Fig. 9h). We hypothesized that the material property of nGFP-NI may cause this solidification. To this end, we performed immunoblotting to differentiate the material properties of specific proteins by analyzing RIPA-lysis-buffer and urea extracts from cells exposed to arsenite stress for 0 to 2 h (Fig. 4g, h and Supplementary Fig. 10a). Surprisingly, although nGFP-NI alone did not localize to SGs in the absence of G3BP1-EGFP, even after two hours of arsenite stress (Supplementary Fig. 3f), both nGFP-NI and nGFP-OGT4 started to appear in the RIPA-insoluble fraction following prolonged stress, with nGFP-NI exhibiting a more rapid aggregation tendency (Fig. 4g). In the presence of G3BP1-EGFP, compared to the nGFP group, the G3BP1-EGFP and nGFP-NI complex also showed increased aggregation in the RIPA-insoluble fraction over longer arsenite treatment, with inclusion of SG markers (eIF4G1 and TDP-43) (Fig. 4h and Supplementary Fig. 10b–d). These findings suggest that the aggregation propensity of nGFP-NI may sequester G3BP1-EGFP into aggregates, thereby inhibiting its LLPS. Collectively, these results indicate a possible dual effect of nGFP-NI: it suppresses the initiation of G3BP1-EGFP LLPS while promoting its solidification, leading to delayed SG formation during short arsenite exposure and the formation of solid-like, irreversible aggregates after prolonged exposure.

nGFP-NI targeted the domain architecture encoded by G3BP1
We next asked whether NI altered the material properties of G3BP1 by targeting any specific domains. G3BP1 contains an N-terminal NTF2L domain responsible for dimerization and two C-terminal RNA-binding regions (RRM and RGG), which are spanned by a long central IDRs (IDR1 and IDR2) (Fig. 5a). These domains collectively enable G3BP1 to act as a molecular switch for LLPS1. Previous studies have underscored the essential roles of G3BP1 dimerization and RNA binding in SG condensation and implied that the long central IDRs only contribute to SG dynamics rather than encoding interactions for SG assembly1,16,19. We observed a consistent inhibitory effect of nGFP-NI on the LLPS of G3BP1 lacking IDR1/2 under arsenite stress (Fig. 5b), suggesting that this inhibition does not rely on IDR1/2. An intriguing feature of the LLPS of G3BP1 is that each of its three major domains can be substituted by heterologous domains with similar properties without disrupting SG assembly1. To test whether nGFP-NI recognizes the precise sequence or a broader structural pattern of G3BP1, we replaced its NTF2L domain with glutathione S-transferase12, a protein that constitutively dimerizes, generating a chimeric protein (GST-G3BP1-EGFP). When coexpressed with nGFP in HeLa cells, GST-G3BP1-EGFP successfully reconstituted SGs under arsenite stress. However, coexpression with nGFP-NI suppressed LLPS of the chimeric protein, despite normal SG formation by endogenous G3BP1 under stress (Fig. 5c). Similarly, we replaced the RNA-binding regions of G3BP1 with tandem RRM domains from an unrelated RNA-binding protein—SRSF4—resulting in the chimeric constructs G3BP1(SRSF4 RRM1/2)-EGFP. The chimera formed SGs normally upon arsenite treatment, yet its LLPS was also blocked by nGFP-NI (Fig. 5d). These domain-swapping experiments suggest that the suppressive effect of nGFP-NI is independent of binding to any specific primary sequence within G3BP1.

Finally, to test this hypothesis, we constructed a synthetic G3BP1 in which all three major domains were replaced by unrelated domains with analogous functions, preserving the modular architecture of the protein (Fig. 5e, f). As expected, this synthetic G3BP1 retained the ability to undergo LLPS and form SGs in the presence of nGFP. However, its arsenite-induced LLPS was still inhibited by nGFP-NI (Fig. 5e, f), further supporting the conclusion that nGFP-NI disrupts G3BP1 LLPS through recognition of a general domain architecture, independent of G3BP1’s exact amino acid sequence.

nGFP-NI is generalizable to other SG components and enables SG regulation
The success of nGFP-NI in targeting synthetic G3BP1 encouraged us to investigate whether this strategy could be generalized to suppress phase separation of other SG components, particularly those with similar modular architecture. We focused on cytosolic SG proteins that contain an oligomerization domain and multiple RBDs, interspersed with IDRs, resembling the structural features of G3BP1. These proteins typically serve as key nodes in assembling complex protein–RNA networks in SGs due to their high valency11,16. Among a panel of abundant and functionally important SG proteins, we selected CAPRIN1, PABPC1, FMR1, and FXR2 for further analysis, all of which have been reported to possess dimerization capability and multiple RBDs16,41,42 (Fig. 6a). As before, we coexpressed each protein with either nGFP or nGFP-NI in HeLa cells. In the nGFP control group, FMR1 and FXR2 formed condensates even before stress, likely due to their high intrinsic phase separation potential (Supplementary Fig. 11a, b), while CAPRIN1 and PABPC1 formed condensates only upon arsenite treatment (Fig. 6b, d). Notably, nGFP-NI suppressed the LLPS of all four proteins, resulting in a marked reduction in the percentage of SG-positive cells (Fig. 6b–f and Supplementary Fig. 11a–c). Similarly, in vitro droplet formation assays with CAPRIN1 and PABPC1 (Supplementary Fig. 11d–f), and cellular assays with FMR1 and FXR2 in U2OS G3BP1/2 dKO cells, further verified the suppressive effect of nGFP-NI (Supplementary Fig. 11g, h). These results also confirm that this effect is determined by direct interaction between the target proteins and nGFP-NI and is independent of the G3BP1-centric protein network. In contrast, SG proteins with distinct domain architectures, such as eIF4G1 and UBAP2L, were not affected by nGFP-NI (Supplementary Fig. 11i, j).

To explore whether the mechanism of LLPS suppression was similar to that observed for G3BP1, we performed FRAP experiments on visible condensates after prolonged arsenite treatment. These analyses revealed that nGFP-NI significantly reduced the fluidity of CAPRIN1, PABPC1, FMR1, and FXR2, resulting in solid-like aggregates and delayed condensate growth (Fig. 6c, e and Supplementary Fig. 11k, l). Together, these findings demonstrate that nGFP-NI can broadly inhibit LLPS across multiple SG proteins, likely by targeting those with a multivalent modular organization similar to G3BP1 and reducing their molecular mobility. Thus, induced proximity between the target protein and the OGT fragment (NI) offers a generalizable strategy for regulating SGs by tuning the material properties of condensates.

Mounting studies revealed close associations of SGs in promoting protein aggregation, halting protein synthesis, as well as disrupting nucleocytoplasmic transport for pathogenesis of several neurodegenerative diseases17,43. Having previously characterized the impact of nGFP-NI on aggregation and translation, we next examined its effect on nucleocytoplasmic transport using a Shuttle-tdTomato reporter system44. This reporter, which contains both a nuclear localization signal (NLS) and a nuclear export signal (NES), normally shuttles from the nucleus to the cytoplasm, and becomes cytoplasmic-enriched during arsenite-induced SG formation—a hallmark of nucleocytoplasmic transport defect. As expected, overexpression of G3BP1-EGFP led to robust cytoplasmic accumulation of Shuttle-tdTomato in response to arsenite stress (Fig. 6g, h). However, when nGFP-NI targeted G3BP1-EGFP to suppress SG formation, the reporter remained largely nuclear, maintaining a nuclear-to-cytoplasmic distribution ratio comparable to unstressed conditions (Fig. 6g, h). This effect mirrors the results observed with other known SG inhibitors44, suggesting that SGs are required for sequestering nucleocytoplasmic transport factors and that disrupting SG formation can mitigate transport defects.

Additionally, since dynamic SGs have been proposed to protect cells during acute stress6, we assessed the effect of nGFP-NI on cell viability following arsenite exposure. Both HeLa and U2OS cells expressing G3BP1-EGFP and nGFP-NI exhibited the lowest survival rates compared to the controls (Fig. 6i and Supplementary Figs. 12 and 13a), indicating that impaired SG dynamics may compromise cellular stress resilience. We also applied nGFP-NI to a cytoplasmically mislocalized TDP43 mutant10, EGFP-TDP-43 NLS mut, and found that nGFP-NI’s targeting significantly reduced cell viability in both HeLa and U2OS cells, likely due to enhanced TDP43 aggregation triggered by nGFP-NI (Supplementary Fig. 13b). Beyond assessing cell viability, we used nGFP-NI to disrupt the co-condensation of G3BP1 and a disease-associated FUS mutant, FUS(R495X)45. When expressed in HeLa cells, FUS(R495X) formed SGs with G3BP1 in over 70% of cells (Supplementary Fig. 13c, d). However, co-expression with nGFP-NI abolished the formation of both FUS(R495X) and G3BP1 condensates. We propose that this failure in granule assembly results from the sequestration of G3BP1 by nGFP-NI. Taking these observations together, given the direct association between the material properties and functions of SGs, this generalizable protein-selective tool holds the potential to investigate and modulate the biological consequences of aberrant SG dynamics of a specific G3BP1-like SG protein.

Methods
Cell culture and transfection
HeLa and U2OS cell lines were cultured in Dulbecco’s Modified Eagle Medium (Gibco, C11995500BT) supplemented with 10% FBS (Gibco, 10099-141C) and 1% penicillin–streptomycin (Gibco, 15140122). HEK-293F cells were cultured in HEK293 Growell TransA (CellPlus Biotechnology, LRU2000). Cells were maintained at 37°C and 5% CO2. TransIT-PRO (Mirus Bio, MIR 5740) and PEI (MedChemExpress, HY-K2014) were used for transient transfections. The U2OS G3BP1-EGFP stable cell line and U2OS G3BP1/2 dKO cell line were described in previous studies44,59.

Plasmids
All plasmids were constructed by cloning PCR-amplified fragments into the pcDNA3.1 vector via homologous recombination (pEASY®-Basic Seamless Cloning and Assembly Kit, TransGen Biotech, CU201), unless otherwise specified. The constructs used in this study—including OGT, OGA, catalytically inactive mutants: OGT(K852A) and OGA(D174N), nanobody fusions: nGFP-OGT4, nGFP-OGT4(K852A) and nGFP-OGA(Stalk), the NLS-tdTomato-NES reporter and EGFP-YTHDF3—were adopted or reproduced from our previous studies27,33,34,44. Variants of OGT (N-Cat, Int-D, NI, and K852A) were generated with a C-terminal 6×His tag. The nGFP-OGT4 series (including truncation mutants ΔTPR, ΔN-Cat, ΔInt-D, and ΔC-Cat) were constructed with a C-terminal HA tag based on the nGFP-OGT4 backbone. SG regulators (eIF4G1, UBAP2L, CAPRIN1, FXR2, FMR1, PABPC1, and RNA-binding domain (SRSF4 RRM1/2) were amplified from their cDNAs and cloned into pcDNA3.1 with an N-terminal FLAG tag, pcDNA3.1_G3BP1-EGFP-FLAG was a gift from Prof. Peng R. Chen (Peking University). Synthetic modules (FRB, FKBP, HaloTag, GST, Ash1) were assembled into pcDNA3.1_G3BP1-EGFP or nGFP-NI constructs. Constructs for protein expression in HeLa cells were validated in Supplementary Fig. 14a. Bacterial expression plasmids (G3BP1-EGFP, EGFP-YTHDF3, EGFP-CAPRIN1, EGFP-PABPC1, OGT, OGT(K852A), and nGFP-NI) were generated using the pET28a vector containing an N-terminal 6×His tag. Lentiviral expression plasmids (nGFP-OGT4, nGFP-ΔNI, and NI) were generated using the pLenti vector containing an N-terminal HA tag. All constructs were verified by Sanger sequencing (Tsingke Biotechnology), and summarized in Supplementary Table 1.

Antibodies and reagents
Commercial antibodies were used under the following conditions: anti-His (Cell Signaling Technology [CST] #12698; 1:400 for immunofluorescence [IF], 1:1000 for immunoblotting [IB]), anti-Myc (CST #2276; 1:1000 IF/IB), anti-OGT (CST #24083; 1:400 IF, 1:1000 IB), anti-HA (Proteintech #66006-2-Ig, 1:400 IF; CST #3724, 1:800 IF, 1:1000 IB), anti-eIF4G (CST #2469; 1:300 IF, 1:1000 IB), anti-UBAP2L (CST #40199; 1:300 IF), anti-G3BP1 (Abcam #ab56574; 1:300 IF, 1:1000 IB), anti-G3BP2 (Abcam #ab86135; 1:300 IF), anti-USP10 (Abcam #ab109219; 1:300 IF), anti-FLAG (CST #14793, 1:800 IF, 1:1000 IB; sigma #F3165, 1:1000 IB), anti-TDP-43 (Proteintech #10782-2-AP; 1:300 IF, 1:1000 IB), anti-peIF2α (CST #9721; 1:1000 IB), anti-eIF2α (CST #5324; 1:1000 IB), anti-puromycin (Millipore #MABE343, 1:1000 IB), anti-O-GlcNAc (RL2) (Abcam #ab2739; 1:1000 IB), anti-ACTIN (Proteintech #66009-1-Ig; 1:10,000 IB), anti-DCP1A (Proteintech #22373-1-AP; 1:400 IF), and anti-SC35 (Abcam #ab11826; 1:300 IF). Secondary antibodies included Alexa Fluor 647/568 conjugates (Thermo #A-21244/#A-11004; 1:1000 IF), IRDye 800CW/680CW (LI-COR #926-32211/#926-68071/#926-32210; 1:10,000 IB), and HRP conjugates: anti-Mouse IgG (HUABIO #HA1006; 1:10,000 IB), anti-Rabbit IgG (HUABIO #HA1001; 1:10,000 IB).

Protein expression and purification
G3BP1-EGFP, EGFP-YTHDF3, EGFP-CAPRIN1, EGFP-PABPC1, and OGT were purified according to previously reported methods1,27,41,60. Briefly, pET28a-based plasmids were transformed into E. coli BL21 (DE3) cells. Single colonies were grown in LB medium with kanamycin (50 μg/mL) at 37°C to OD600 0.6–0.8, then induced with 0.5 mM IPTG at 25°C for 16 h (G3BP1-EGFP and EGFP-YTHDF3) or 16°C for 16 h (EGFP-CAPRIN1, EGFP-PABPC1, OGT, OGT(K852A), and nGFP-NI). For nGFP production, HEK293F cells were transfected with corresponding plasmids using PEI and cultured for 96 h.

Bacterial cells were harvested by centrifugation (4000×g, 20 min, 4°C), resuspended in lysis buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1× protease inhibitors), and lysed by sonication (150 W, 3s on/5s off, ice-cooled, 30 min total). Lysates were clarified by centrifugation (12,000×g, 30 min, 4°C). For G3BP1-EGFP, EGFP-YTHDF3, EGFP-CAPRIN1, EGFP-PABPC1, OGT, and OGT(K852A), supernatants were loaded onto Ni-NTA columns pre-equilibrated with lysis buffer, washed with buffer containing 80 mM imidazole, and eluted with 200 mM imidazole. Eluted proteins were concentrated using 10K/30K Amicon spin columns (Merck-Millipore, UFC901096/ UFC903096) (4000×g, 30 min, 4°C) and buffer-exchanged into storage buffers (G3BP1-EGFP and EGFP-CAPRIN1: 20 mM Tris-HCl pH 7.5, 400 mM NaCl, 0.5 mM DTT, 10% glycerol; EGFP-YTHDF3: 20 mM HEPES pH 7.4, 300 mM KCl, 6 mM MgCl2, 0.02% NP-40, 20% glycerol; EGFP-PABPC1: 5 mM Tris pH 7.4, 250 mM NaCl, 0.25 mM TCEP, 10% glycerol; OGT and OGT(K852A): 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5 mM EDTA, 2 mM DTT, 20% glycerol).

For nGFP-NI, after initial centrifugation and lysis, the insoluble pellet was resuspended in PBS-Urea buffer (PBS, 8 M urea, pH 7.4), recentrifuged (12,000×g, 20 min, 4°C), and the supernatant loaded onto a Ni-NTA column. The column was washed with PBS-Urea buffer containing 50 mM imidazole and eluted with 500 mM imidazole. The eluate was further purified by a Superdex™ 200 Increase 10/300 GL column (Cytiva, cat. 28990944). The column was equilibrated with storage buffer (PBS, 300 mM NaCl, 10% glycerol, pH 7.4) and fractions containing nGFP-NI were collected.

HEK293F cell supernatants (for nGFP) were collected by centrifugation (10,000×g, 10 min, 4°C) and purified using Ni-NTA chromatography as described for bacterial proteins. Purified nGFP was buffer-exchanged into PBS with 10% glycerol (pH 7.4). Protein concentrations were quantified via NanoDrop One spectrophotometer (Thermo) and stored at −80°C. The Coomassie-stained gel image of the above purified proteins is shown in Supplementary Fig. 14b.

Immunofluorescence and microscopy
Cells were seeded into 4-well confocal dishes (JingAn Biological, J40204) and exposed to indicated stress conditions. Post-treatment, cells were fixed with 4% paraformaldehyde (Leagene, DF0134) for 15 min, washed three times with PBS, and blocked for 1 h at room temperature in PBS containing 5% BSA and 0.3% Triton X-100. Cells were then incubated sequentially with primary and secondary antibodies (diluted in 1% BSA/0.3% Triton X-100/PBS) for 1 h each at room temperature, with three PBS washes between steps. Nuclei were counterstained with DAPI (Thermo, R37606) followed by three PBS washes. Imaging was performed using a Dragonfly spinning-disk confocal microscope (Andor Technology, CR-DFLY-202-2540).

Immunoblotting
Cells were washed with PBS and lysed on ice for 30 min in RIPA buffer (Applygen, C1053-500) containing 1× EDTA-free protease inhibitor cocktail (MCE, HY-K0010). Lysates were clarified by centrifugation (12,000×g, 20 min, 4°C), and supernatants were mixed with 1× SDS buffer, followed by denaturation at 95°C for 5 min. Proteins were separated on 4–12% Bis-Tris gradient gels (Genscript, M00654) and transferred to nitrocellulose membranes (Millipore, HATF00010). Membranes were blocked with 5% non-fat milk/TBST for 1 h at room temperature, then sequentially incubated with primary and secondary antibodies for 1 h each at room temperature, with three 1× TBST washes between steps. Signals were visualized using an Azure Imager C600 (Azure Biosystems).

Immunoprecipitation
HeLa cells were cultured to >90% confluency in 6-well plates, harvested, and washed with PBS. Cells were lysed in RIPA buffer supplemented with 1× EDTA-free protease inhibitor cocktail, incubated on ice for 20 min, and clarified by centrifugation (12,000×g, 10 min, 4°C). Supernatants were incubated with anti-FLAG (Selleck, B26101; Sigma, M8823) or anti-HA affinity beads (Selleck, B26201) at 4°C overnight under gentle rotation. Beads were washed thrice with 500 µL PBST, and immunoprecipitated complexes were eluted by boiling in 1× SDS buffer (95°C, 5 min). Eluted proteins were resolved via SDS-PAGE, transferred to nitrocellulose membranes, and analyzed by immunoblotting.

Liquid-liquid phase separation
G3BP1-EGFP, nGFP, nGFP-NI, and OGT were buffer-exchanged into LLPS buffer (150 mM NaCl, 50 mM HEPES pH 7.5) using 10/30 kDa MWCO filters (Merck-Millipore, UFC5010/5030), while EGFP-YTHDF3 was buffer-exchanged into PBS via the same method. OGT, nGFP, and nGFP-NI were Cy5-labeled via 4 h room-temperature incubation. G3BP1-EGFP phase separation was induced by adding HeLa total RNA (Yeasen, 19231ES) or 10% PEG8000 (Coolaber, CP8241). Samples were mixed, loaded into a custom imaging chamber (glass coverslip adhered to a slide with double-sided tape, ~10 µL volume), and allowed to phase separate for 3 min. Droplets were imaged using a Dragonfly spinning-disk confocal microscope (Andor Technology, CR-DFLY-202-2540) with a 20× or 63× objective, and images were analyzed in Imaris Viewer (v10.2.0) and ImageJ.

Fluorescence recovery after photobleaching (FRAP)
For FRAP assays, in vitro phase-separated droplets and cellular condensates were analyzed as follows: In vitro samples were prepared as described previously, while cellular experiments involved seeding cells in 4-well confocal dishes, transfecting with target plasmids, and applying stress treatments 24 h post-transfection. Live-cell imaging was performed under physiological conditions (37°C, 5% CO2) using a ZEISS LSM900 microscope equipped with a live-cell system. Target condensates were photobleached with a 488 nm laser (80% intensity), and fluorescence recovery was monitored at defined intervals. Raw intensity data were normalized in ImageJ, with pre-bleach signals set to 100% and recovery expressed as relative percentages of initial intensity to calculate dynamic exchange rates.

In vitro glycosylation assay
Reaction mixtures (10 μM target protein, 1 μM OGT, 1 mM UDP-GlcNAc [Millipore, U4375]) were prepared in reaction buffer (25 mM Tris pH 7.5, 50 mM NaCl, 0.5 mM EDTA, 2 mM DTT, 10% glycerol) and incubated at 30°C for 4 h with 200 rpm agitation. Post-reaction, samples were denatured in 1× SDS buffer (95°C, 5 min), resolved by SDS-PAGE, and subjected to immunoblotting detected by indicated antibodies.

Separation of RIPA lysis buffer-soluble and insoluble fraction
According to a previously reported method61 with minor modifications, stress granule isolation was performed at 4°C. Briefly, cells were washed once with PBS and processed in parallel: (1) For total lysates, cells were lysed in Urea buffer (8 M urea, 2% SDS, 50 mM Tris-HCl pH 7.4), mixed with 1× SDS buffer. (2) For fractionation, cells were lysed in RIPA buffer (with 1× EDTA-free protease inhibitor cocktail) on ice for 30 min, sequentially centrifuged (4000×g, 5 min; 18,000×g, 20 min) to collect the soluble fraction (S), which was denatured as above. The insoluble pellet was washed with RIPA buffer (18,000×g, 5 min), resolubilized in Urea buffer, and denatured to generate the pellet fraction (P). All samples were resolved by SDS-PAGE for immunoblotting.

Puromycin incorporation assay
The puromycin incorporation assay was modified from Kedersha et al.62. HeLa cells were treated with or without 500 μM NaAsO2 for 30 min and treated with 10 µg/mL puromycin (Gibco, A1138-03) for 15 min prior to lysis. Whole-cell lysates were analyzed by SDS-PAGE followed by Coomassie staining or Western Blot using an anti-puromycin antibody.

Cell viability
Cell viability was assessed 24 h post-transfection by inducing stress with 500 μM NaAsO2 for an additional 24 h. Cells were harvested (500×g, 3 min), washed twice with PBS, and resuspended in 500 μl PBS. Propidium iodide (PI) (Biosharp, BL708A) was added (1 μg/mL final) and incubated in the dark at room temperature for 20 min to label dead/damaged cells. GFP fluorescence (transfection marker) and PI emission (excitation: 488 nm; emission: 617 nm) were simultaneously analyzed via flow cytometry (Thermo, Attune NxT). Live (GFP+/PI−) and dead (GFP+/PI+) cells were gated based on fluorescence intensity, with data processed using FlowJo.

MTT assay
Cell viability was assessed via the rapid MTT assay after seeding cells in 96-well plates. Culture medium was replaced with 100 µL fresh medium, followed by 10 µL of 12 mM MTT stock (MedChemExpress, HY-15924) per well; medium-only wells served as negative controls. Following 37°C incubation for 2–5 h, medium was aspirated, and 50 µL DMSO was added to dissolve formazan crystals. Plates were further incubated at 37°C for 10 min, thoroughly mixed, and absorbance measured at 540 nm.

Cross-linking of recombinant proteins and MS analysis
In a 100 μL reaction volume, 6 μM G3BP1-EGFP was incubated with 2 μM nGFP or nGFP-NI and 100 μM BS3 (Thermo, 21595) in reaction buffer (400 mM NaCl, 50 mM HEPES, pH 7.5) at room temperature for 30 min, then quenched with 50 mM Tris (pH 7.5) for 15 min. Proteins were precipitated with methanol/chloroform, centrifuged at 15,000×g for 10 min, and the pellets were washed twice with methanol before resuspension in 6 M urea. After adding 10 mM TCEP (Sigma, C4706) and 20 mM iodoacetamide (Sigma, I6125) at 37°C for 30 min, 1× TEAB (Sigma, T7408) was added to dilute urea to 1 M, followed by trypsin (Promega, V5111) for enzymatic digestion at 37°C with rotation for 16 h. Resulting peptides were dried by vacuum centrifugation, fractionated using Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo, 84868), and finally resuspended in 0.1% formic acid (FA) for MS analysis. XL-MS analysis was performed once.

Tandem MS
LC−MS/MS analysis was performed using an UltiMateTM 3000 RSLCnano System (Thermo) coupled to an Orbitrap Exploris 480 equipped with a nanospray flex ion source (Thermo). Mobile phases A and B were water with 0.1% (v/v) FA and 80% acetonitrile with 0.1% (v/v) FA. Peptides were separated using a linear gradient from 10% to 28% B within 45 min, followed by an increase to 32% B within 6 min and further to 99% B within 2 min and re-equilibration. The MS1 spectra (350–1500 m/z) were obtained at a resolution of 60,000, an AGC target of 3×106 and a maximum injection time of 50 ms. HCD fragmentation was performed on the most abundant precursors exhibiting a charge state from 2 to 6 of an AGC target of 1×105 by isolating them in the quadrupole mode at 1.6 m/z and with fragmentation energy of 30%.

MS data analysis
Crosslinked peptides were identified using pLink2 software (v. 2.3.9) with the following search parameters: precursor mass tolerance 10 ppm, fragment mass tolerance 20 ppm, peptide length per chain 6–60 amino acids, peptide mass per chain 600–6000 Da, variable modification of Cys (+57.02146), trypsin as the enzyme, and up to 3 missed cleavages per chain. For crosslinked samples, searches were performed against FASTA files containing sequence information of nGFP, nGFP-NI, and G3BP1-EGFP.

Statistics and reproducibility
Colocalization analysis was performed using the Fiji (version 2.9.0). GraphPad Prism 9 was used for statistical analysis. Data are reported as mean±s.d. or mean±s.e.m. Normality (Shapiro–Wilk test) and homogeneity of variances (Brown–Forsythe test) were checked before choosing the statistical test. Two-group comparisons used unpaired two-tailed Student’s t-test (parametric) or Mann–Whitney U test (non-parametric), while >2 groups were analysed by Welch’s one-way ANOVA with Games–Howell post-hoc (parametric) or Kruskal–Wallis with Dunn’s multiple-comparison test (non-parametric). All P values were obtained from GraphPad Prism 9. P<0.05 was considered to be statistically significant. For extremely significant results (e.g., P<1×10−15), the software restricts output to 15 decimal places; in these cases, we report the smallest displayable value to ensure transparency and reproducibility. The sample size (n) represents biological replicates per experiment.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
Unless otherwise stated, all data supporting the results of this study can be found in the article, supplementary, and source data files. Source Data are provided with this paper. The XL-MS data have been deposited to the ProteomeXchange Consortium through the iProX63,64 repository with the dataset identifier PXD068817 [https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD068817]. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Wang et al. report on the modulation of liquid-liquid phase separation (LLPS) of proteins like G3BP1 to form Stress Granules (SGs) using a repurposed O-GlcNAc transferase (OGT) in their manuscript titled \"Targeted stress granule regulation by engineering a non-catalytic O-GlcNAc transferase\". The authors demonstrate that by chemical design induced proximity of the protein and OGT, the LLPS propensity of the protein is effectively suppressed, leading to formation of smaller/fewer SGs irrespective of the catalytic activity of the OGT. Using deletion variants of the OGT, the authors identify that the N-Cat and Int-D segments are crucial for this suppression activity and consequently design a nGFP-NI construct that if most effective at arresting SG formation via LLPS. The LLPS modulation is determined by the order of addition of the nGFP-NI with respect to the stressor (arsenite) and leads to changes in the material properties of the condensates, with increased solid-like physical characteristics. The generality of such a construct in inhibiting SG formation for similar classes of proteins is demonstrated by using engineered variants of G3BP1 as well as a synthetic protein. Overall, the authors have demonstrated the versatility of the approach to modulating LLPS of proteins/RNA to form SGs using OGT-derived nGFP-NI. The control over SG formation is an important problem and therefore this manuscript is of interest to the community. However, prior to being considered for publication, the authors should address the following concerns -\n1) A primary concern of mine is that the inability of the nGFP-NI to reverse pre-formed SGs in Figure 5 and the importance of stressor treatment after pre-incubation with the nGFP-NI (Figure 3 and extended data Figure 3 c-d) is primarily due to the change of state for the SG condensate. The FRAP data in Figure 4 d-e and h-i indicates the formation of gel-like or solid-like phase post condensation, with low dynamics. Therefore, the addition of nGFP-NI is unable to partition into and interact with the aggregated G3BP1 and cause the disassembly/shrinkage of the SGs. This warrants some discussion in the manuscript as the Figure 3 discussion precedes the insight about changes to the liquid-like SG in Figure 4.\n2) The immobile fraction in the HeLa cells in Figure 4 e is substantially higher at ~ 80% vs. the in vitro experiment in Figure 4 h-i at ~ 40% when incubated with nGFP-NI. What could be the cause for such a striking difference in the change in viscosity/material-state of the SGs in-vivo vs. in-vitro?\n3) The generalizability of the approach to CAPRIN1 and PABPC1 as components of SGs is interesting, however it appears that the components themselves have a higher immobile fraction in the nGFP control at ~40%. In Extended Data Figure 5, similarly, FMR1 and FXR2 have highly solid-like SGs (60% immobile fraction). Does the ability of the the nGFP-NI to arrest condensation into SGs get influenced by the physical properties of the condensate components?\n4) Visually, the SGs formed by RNA complexation and PEG8000 molecular crowding appear to have different droplet size distribution in Extended Data Figure 3 e-f. Is the nGFP-NI system more effective at LLPS suppression in presence one of the above mentioned triggers?", "reviewer_2": "The manuscript by Wang et al. reports a new strategy for modulating stress granules (SGs) by engineering a non-catalytic function of O-GlcNAc transferase (OGT). The authors identify that OGT suppresses G3BP1-driven liquid–liquid phase separation (LLPS) and SG assembly in an enzymatically independent manner. They further design a truncated OGT construct (termed “NI”) and develop a modular platform, “STOP”, which employs nanobody- or chemically induced proximity to inhibit SG protein condensation by reducing molecular mobility. The study is technically rigorous and conceptually innovative. STOP is introduced as a genetically encoded, generalizable tool for condensate manipulation, and the work sheds light on the previously underappreciated functional role of OGT’s intervening domain (Int-D). The authors validate STOP across multiple SG proteins and demonstrate its utility in perturbing SG dynamics during cellular stress.\nHowever, several mechanistic and conceptual issues remain to be addressed. These include the lack of direct mechanistic evidence for NI function, uncertainties regarding STOP’s specificity and generalizability, and the limited physiological validation using endogenous systems. Further clarification and experimentation in these areas would significantly enhance the impact and robustness of the study.\n1. While the authors show that the NI module inhibits G3BP1 LLPS and SG formation, the mechanism remains poorly defined. The proposed mode of action—valence capping or interference with multivalent interactions—is not supported by direct biochemical or structural evidence. Furthermore, the observation that nGFP-NI promotes G3BP1 aggregation under prolonged stress appears inconsistent with the notion of reduced valency, suggesting a possible dual effect: inhibition of LLPS initiation but promotion of solidification. The biological consequences of this phase transition are also underexplored. Given that SG solidification is associated with neurodegenerative disease pathology, further studies on the cellular impact, reversibility, and potential toxicity of STOP-induced aggregates are necessary.\n2. The authors propose STOP as a general tool for targeting SG components, based on its suppression of condensation in proteins such as CAPRIN1, FMR1, and PABPC1. However, it is unclear whether these effects arise from direct interactions with each protein or from indirect network-level perturbation via G3BP1 inhibition. Additionally, potential off-target effects on other membraneless organelles (e.g., P-bodies, nucleoli, nuclear speckles) are not assessed. Testing STOP in systems devoid of G3BP1, performing direct binding or co-localization assays, and evaluating its effects on unrelated condensates would help clarify its specificity and broaden the scope of its applicability.\n3. The majority of the experiments rely on overexpression of tagged G3BP1 constructs (e.g., G3BP1-EGFP or Halo-tagged variants), which may not reflect physiological stoichiometry or native dynamics. This raises the possibility of artifacts in LLPS behavior or STOP-induced suppression. The efficacy of STOP in modulating SGs under endogenous protein levels remains unclear. The authors are encouraged to test STOP function in CRISPR knock-in cell lines expressing endogenous tagged G3BP1, or via complementation assays in G3BP1/2 knockout cells. Quantitative comparison of STOP versus endogenous protein expression would also be informative.", "reviewer_3": "The manuscript by Wang et al. proposes a previously uncharacterized non-catalytic function of human OGT where a fragment covering the N-terminal catalytic lobe and intervening domain (NI) of OGT appears to delay stress granule (SG) formation under sodium arsenite-induced stress. Based on this, the authors developed a modular tool that induces proximity of the OGT-NI domain to G3BP1 and other SG-nucleating proteins sharing similar domain architectures, thereby modulating SG formation. Mechanistically, the authors suggest that this modulation is achieved by altering the material properties of SGs, promoting more solid-like aggregates rather than liquid-like condensates.\nWhile this study potentially identifies a novel (non-catalytic role) of OGT in modulating stress granule formation and introduces a promixity-inducing approach (albeit relying on existing tools/approaches) there are significants flaws that make the solidity of the conclusions drawn questionnable and therefore this is not a reliable advance of scientific knowledge.\nMajor concerns\n1) Physiological relevance. The findings rely heavily on 1) overexpression, 2) a system that forces OGT to the stress granules where (even according to the authors’ own experiments) it does not occur/belong and 3) the use of a carcinogenic cell line (HeLa) with questionnable physiological relevance. It remains unclear whether the OGT-NI domain has a physiological role in SG modulation under endogenous expression levels. Does the NI domain delay SG formation without overexpression? What happens in a stable cell line carrying point mutations or deletion in the specific OGT domains?\n2) Producing an isolated ND-NI fragment is a blunt tool. The N-terminal and intervening domains of OGT are not at all “domains”. They are closely packed forming a continuous catalytic domain. Chopping them up like this will exposed a hydrophobic core, producing insoluble protein, likely to precipitate, for instance, in/on stress granules. Nowhere do the authors show that this is a bona fide soluble protein in an Eppendorf tube when produced in vitro.\n3) Biological Utility of the Tool. Although the tool delays SG formation, it also promotes solid-like G3BP1 aggregation. How beneficial is this in the broader context of SG biology? Would this perturbation itself have deleterious consequences for cell physiology?\n4) Context-Specificity of Discovery. The role of OGT-NI in SG regulation appears limited to G3BP1 or G3BP1-like proteins under arsenite stress. However, other SG-forming proteins (e.g., UBAP2L, eIF4G1) contribute to SG assembly under different stress conditions (e.g., osmotic, ER stress). The extent to which this mechanism generalizes remains unclear.\nSpecific comments:\nIn the title, text and discussion, authors strongly highlight that the effect on stress granules is independent of OGT’s catalytic activity, while most of the construct used still includes part of the catalytic domain.\nThe interaction between OGT and G3BP1 is strongly dependent on the affinity between nGFP and EGFP. Is this mechanism even possible in vivo or is it a forced interaction?\nFigure 1: 60% normal SG formation vs 40% reduced SG. The authors should comment on that.\nExtended Fig. 1b:\n1. Why is there a FLAG tag band in lane 1, despite no protein being expressed?\n2. Why are HIS-tag bands present in lanes 1 and 8?\n3. Why are there no OGT bands in lanes 5–8 and 12–13, where OGT(K842A) was used? Why did they use this specific mutation (the rest of the field tends to use the isosteric K842M mutation)?\n4. Why does the HIS tag disappear in lane 4?\n5. Extended Fig. 1b vs. 1c: These panels seem contradictory. In 1b, OGT does not appear to interact with G3BP1, whereas in 1c, OGT co-immunoprecipitates with G3BP1. Please clarify this discrepancy.\nFigure 2b: What is the sequence of the OGA(stalk) construct used?\nExtended Fig. 2a / Line 158-162: The HA-nGFP-NI construct is used in Fig. 2a before it is introduced in the main text (line 162). Please reorder to avoid confusion.\nExtended Fig. 2e: The co-localization of G3BP1 with eIF4G1 under sorbitol-induced stress appears different from UBAP2L. Please comment on this observation.\nFigure 3e: The interaction of the truncated versions of OGT is forced with the nanobody/GFP. What happens when the interaction is not forced (like in Figure 1g)?\nExtended Fig. 3: The figure labelling does not match the legend. Please revise for consistency.\nExtended Fig. 3b: At 0 µM rapamycin, HA signal is observed in FLAG pull-down, suggesting baseline interaction. Does this impact the interpretation of the results?\nData analysis of some data (like granule area) shouldn't have been done with a parametric test, since data is not normally distributed.\nLine 52: Please explain or reference what “NTF2L-targeting” refers to.\nLine 53: Sentence lacks a reference.\nLine 55: Why can’t existing regulatory tools for G3BP1/2 be adapted to other SG proteins?\nLine 97: “We synthesized and applied a chimeric molecule (…)” The phrasing suggest that the molecule is a new creation – this is not the case. Rephrasing as “resynthesized” would be more precise.\nLine 186: “Rapamycin treatment without inducing observable cellular stress”. Please elaborate. What indicators were assessed to conclude that there was no observable stress?\nLine 210: The text suggests that the phenotype “may not stem from disruption of G3BP1’s RNA-binding capacity.” Please provide more evidence or rationale to support this claim.\nIn line 222, the authors prove that SG assembly proceeds normally prior to G3BP1 involvement, while in line 226 they want to prove that nGFP-NI prevents or delays SG assembly. Some parts of the manuscript should be written more clearly.\nLine 231: The text refers to “sequestering TDP-43 together,” but TDP-43 is not observable in Extended Fig. 4c.\nLine 233 / Fig. 4a: The statement claims that puncta in nGFP-NI-expressing cells are more aggregated, but Fig. 4a seems to show this phenotype in nGFP-only expressing cells. Please verify.\nLine 252 / Extended Fig. 4j: The claim about increased eIF4G1 inclusion is not supported, as no eIF4G1 bands are visible in the figure."}, "author_response": "Response: We are grateful to the reviewer’s appreciation on the importance of our study, as well as their useful comments which guided us to a significantly strengthened manuscript. The point-by-point response is provided below.\nComment 1: 1) A primary concern of mine is that the inability of the nGFP-NI to reverse pre-formed SGs in Figure 5 and the importance of stressor treatment after pre-incubation with the nGFP-NI (Figure 3 and extended data Figure 3 c-d) is primarily due to the change of state for the SG condensate. The FRAP data in Figure 4 d-e and h-i indicates the formation of gel-like or solid-like phase post condensation, with low dynamics. Therefore, the addition of nGFP-NI is unable to partition into and interact with the aggregated G3BP1 and cause the disassembly/shrinkage of the SGs. This warrants some discussion in the manuscript as the Figure 3 discussion precedes the insight about changes to the liquid-like SG in Figure 4.\nResponse 1: Thank you for this suggestion. As the Reviewer suggested, we re-organized the “nGFP-NI suppresses G3BP1 LLPS in an order- and concentration-dependent 1 manner” section and the “nGFP-NI promoted G3BP1 aggregation” section, and added more discussion (please see the highlighted text).\nHere we would also like to clarify the results in Fig. 3 and Fig. 4 to resolve any potential confusion. The data shown in Fig. 3 are intended to demonstrate that nGFP-NI prevents the normal process of G3BP1 LLPS both in cells and in vitro, rather than dissolving pre-formed condensates. In the experiments shown in Fig. 3c and the top panels of Fig. 3e-f, nGFP-NI is still able to partition into G3BP1 condensates due to the strong GFP–nGFP interaction. However, because G3BP1 undergoes a conformational change and its protein network is already established after arsenite stress, we postulate that recruiting nGFP-NI after condensate formation does not disrupt the existing network and is therefore unable to disassemble the droplets. The data in Fig. 4 suggest that normal stress granules exhibit dynamic, liquid-like properties, whereas nGFP-NI treatment results in a more gel-like phase. We performed additional experiments to explore the underlying mechanism and have included these new data in Fig. 4g. These results indicate that nGFP-NI tends to aggregate under prolonged arsenite stress, which may sequester its target protein into a more aggregated state. Further interpretation of these findings and the proposed mechanism can be found in Response 1 to Reviewer #2 Comment 1.\nComment 2: 2) The immobile fraction in the HeLa cells in Figure 4 e is substantially higher at ~ 80% vs. the in vitro experiment in Figure 4 h-i at ~ 40% when incubated with nGFP-NI. What could be the cause for such a striking difference in the change in viscosity/material-state of the SGs in-vivo vs. in-vitro?\nResponse 2: We speculate that the observed difference may arise from distinct experimental conditions: 1) time difference: in vivo data were acquired after 2 hours of 500 μM NaAsO₂ treatment (Fig. 4e), whereas in vitro droplets were analyzed approximately 5 minutes after the addition of total RNA or PEG8000 (now listed as Supplementary Fig. 8g); 2) environment difference: in vivo experiments occur in a cellular environment containing thousands of proteins, diverse RNA species, and other regulatory factors, which collectively form a complicated network for granule formation. In contrast, the in vitro system consists solely of G3BP1-EGFP, nGFP-NI, and total RNA in LLPS buffer (150 mM NaCl, 50 mM HEPES pH 7.5). These differences likely account for the discrepancy in the immobile fraction of G3BP1 condensates observed in cells versus in vitro.\nComment 3: 3) The generalizability of the approach to CAPRIN1 and PABPC1 as components of SGs is interesting, however it appears that the components themselves have a higher immobile fraction in the nGFP control at ~40%. In Extended Data Figure 5, similarly, FMR1 and FXR2 have highly solid-like SGs (60% immobile fraction). Does the ability of the the nGFP-NI to arrest condensation into SGs get influenced by the physical properties of the condensate components?\nResponse 3: As the reviewer noticed, these SG components exhibit different LLPS capabilities. In the four cases examined in our study, nGFP-NI effectively sequestered and suppressed the LLPS of all four proteins. Furthermore, we used YTHDF3—another phase-separating protein—as a negative control for nGFP-NI. YTHDF3 also exhibits a high immobile fraction (~60%) (Chen et al. Nat. Cell Biol. 25, 1676-1690 (2023)), yet it was not suppressed by nGFP-NI. We therefore conclude that the efficacy of nGFP-NI is not broadly correlated with the physical properties of target proteins, at least under the conditions tested here. Instead, as illustrated in Fig. 5 and Fig. 6a–f, the domain architecture of the target protein appears to be a more critical determinant of nGFP-NI’s effect.\nComment 4: 4) Visually, the SGs formed by RNA complexation and PEG8000 molecular crowding appear to have different droplet size distribution in Extended Data Figure 3 e-f. Is the nGFP-NI system more effective at LLPS suppression in presence one of the above mentioned triggers?\nResponse 4: The observed differences in droplet size likely stem from the distinct mechanisms by which RNA and the crowding agent PEG8000 induce G3BP1 LLPS—a phenomenon commonly observed with other phase-separating proteins. As shown in Fig. 3e-f, nGFP-NI is effective under both conditions. In the presence of RNA, nGFP-NI reduced the average G3BP1 droplet size from 42.66 µm2 to 26.31 µm2; with PEG8000, the reduction was from 6.17 µm2 to 2.62 µm2. In both cases, this represents an approximately 2-fold decrease.\nThe manuscript by Wang et al. reports a new strategy for modulating stress granules (SGs) by engineering a non-catalytic function of O-GlcNAc transferase (OGT). The authors identify that OGT suppresses G3BP1-driven liquid–liquid phase separation (LLPS) and SG assembly in an enzymatically independent manner. They further design a truncated OGT construct (termed “NI”) and develop a modular platform, “STOP”, which employs nanobody- or chemically induced proximity to inhibit SG protein condensation by reducing molecular mobility. The study is technically rigorous and conceptually innovative. STOP is introduced as a genetically encoded, generalizable tool for condensate manipulation, and the work sheds light on the previously underappreciated functional role of OGT’s intervening domain (Int-D). The authors validate STOP across multiple SG proteins and demonstrate its utility in perturbing SG dynamics during cellular stress.\nHowever, several mechanistic and conceptual issues remain to be addressed. These include the lack of direct mechanistic evidence for NI function, uncertainties regarding STOP’s specificity and generalizability, and the limited physiological validation using endogenous systems. Further clarification and experimentation in these areas would significantly enhance the impact and robustness of the study.\nResponse: We thank the reviewer for their positive assessment of our study's rigor and innovation. By leveraging the unique noncatalytic functions of OGT combined with induced proximity approaches, we present STOP (nGFP-NI) as a general tool for suppressing the LLPS of G3BP1 and four other stress granule components.\nTo fully address the reviewer’s concerns, we have incorporated additional in vitro and in vivo evidence as outlined below:\n1) We performed cross-linking mass spectrometry and aggregate separation assays to elucidate the potential mechanism of nGFP-NI action.\n2) We validated the direct effect of nGFP-NI on target proteins by applying it to purified CAPRIN1 and PABPC1 in vitro, and to FXR2 and FMR1 in U2OS G3BP1/2 double knockout (dKO) cells.\n3) We evaluated the specificity of nGFP-NI by examining its effect on other biomolecular condensates under identical conditions.\n4) We included further validation in U2OS cells stably expressing G3BP1-EGFP, as well as in U2OS G3BP1/2 dKO cells.\n5) We added data demonstrating the biological consequences of nGFP-NI-induced SG suppression, including effects on cell viability and disease-related protein condensate formation.\nWe elaborate on these points in the detailed response that follows.\nComment 1: 1. While the authors show that the NI module inhibits G3BP1 LLPS and SG formation, the mechanism remains poorly defined. The proposed mode of action—valence capping or interference with multivalent interactions—is not supported by direct biochemical or structural evidence. Furthermore, the observation that nGFP-NI promotes G3BP1 aggregation under prolonged stress appears inconsistent with the notion of reduced valency, suggesting a possible dual effect: inhibition of LLPS initiation but promotion of solidification.\nResponse 1: As the reviewer suggested, we further investigated the underlying mechanism through two key experiments. We greatly appreciate the reviewer's insight regarding a potential dual effect, with which we fully agree.\nFirst, given the challenge of obtaining reliable structural resolution or prediction for IDR-containing proteins, we performed chemical cross-linking on complexes of G3BP1-EGFP with either nGFP or nGFP-NI and analyzed their interactions by mass spectrometry (new Supplementary Fig. 7). In the G3BP1-EGFP/nGFP complex, cross-links were primarily observed between two structural regions: the N-terminal NTF2-like (NTF2L) domain and the C-terminal RNA-binding region (RRM and RGG). This is consistent with previous reports that G3BP1 adopts a closed conformation, with the basic C-terminal RGG bending back to interact with the N-terminal region prior to LLPS induction (Yang, P. et al. Cell 181, 325-345 (2020)). In contrast, after pre-incubation with nGFP-NI, most intramolecular cross-links disappeared, and two new cross-links between G3BP1-EGFP and nGFP-NI were detected. The limited number of cross-links is attributable to the scarcity of accessible lysine residues in the two constructs suitable for crosslinking. These results suggest that while nGFP alone does not alter G3BP1’s intramolecular interactions, nGFP-NI may sequester G3BP1-EGFP prior to the addition of LLPS inducers— corresponding to the “inhibition of LLPS initiation” proposed by the reviewer. These findings also partially support our data in Fig. 5, indicating that nGFP-NI may simultaneously engage multiple domains of G3BP1. These results have been included as Supplementary Fig. 7 in the revised manuscript.\nNext, we fractionated RIPA-soluble and insoluble lysates from HeLa cells expressing nGFP-OGT4 or nGFP-NI after exposure to arsenite stress for varying durations (new Fig. 4g). Notably, although most proteins—including endogenous G3BP1—remained soluble in RIPA buffer, nGFP-NI was detected in the insoluble fraction as early as 0.5 hours post- stress, while nGFP-OGT4 also became insoluble after 2 hours. These results indicate that nGFP-OGT4, typically regarded as a soluble protein, can form aggregates rather than liquid-like stress granules under prolonged stress. Moreover, nGFP-NI—a truncation of nGFP-OGT4—exhibits an even greater propensity for rapid aggregation. Integrating these findings with data in Fig. 4h and Supplementary Fig. 9, we propose a mechanism for nGFP-NI–mediated SG suppression: by targeting G3BP1-EGFP, nGFP-NI sequesters it into insoluble aggregates, thereby inhibiting LLPS initiation initially and promoting solidification over time. The net phenotypic outcome likely reflects a balance between the target protein’s LLPS propensity and nGFP-NI’s aggregation tendency. In accordance with the reviewer’s suggestion, we have revised the “nGFP-NI promoted G3BP1 aggregation” section to incorporate this “dual-effect” model. These new results are presented in Fig. 4g.\nThe biological consequences of this phase transition are also underexplored. Given that SG solidification is associated with neurodegenerative disease pathology, further studies on the cellular impact, reversibility, and potential toxicity of STOP-induced aggregates are necessary.\nWe agree with the reviewer on the importance of understanding the biological consequences of this phase transition. Although stress granules are often studied in the context of neurodegenerative diseases, they also play critical roles in other pathological conditions such as cancer, viral infection, and cardiovascular disease (Gao, X. et al. Br. J. Pharmacol. 176, 4421-4433 (2019); Grabocka, E. & Bar-Sagi, D. Cell 167, 1803-1813 (2016); Jayabalan, A.K., Griffin, D.E. & Leung, A.K.L. Viruses 15, 449 (2023); Panas, M.D. et al. PLoS Pathog. 11, e1004659 (2015); Yang, G. et al. Cardiovasc. Transl. Res. 18, 738-748 (2025)). In our previous manuscript version, we demonstrated how nGFP-NI-induced disruption of G3BP1 LLPS affects nucleocytoplasmic transport (Fig. 6g,h) and cell viability (Fig. 6i), highlighting the tool's potential for perturbing SG function. During revision, we further investigated whether nGFP-NI–mediated targeting of G3BP1 and G3BP1 influences granules containing disease-associated proteins. Perturbation of granules formed by a disease-related TDP-43 mutant with nGFP-NI similarly reduced cell viability, suggesting that TDP-43 solidification accelerates cell death under stress (new Supplementary Fig. 11b). These results reinforce the established concept that disrupting normal liquid-like SG dynamics can impair viability—as reported in other studies (Johnson, B.S. et al. J. Biol. Chem. 284, 20329-20339 (2009); Protter DSW, Parker R. Trends. Cell Biol. 26, 668-679 (2016))—and underscore the general applicability of nGFP-NI, as further supported by Fig. 6a–f.\nIn addition, we co-transfected the disease-associated mutant FUS(R495X) together with G3BP1-EGFP and nGFP or nGFP-NI into HeLa cells according to previous studies (Dormann, D. et al. EMBO J. 29, 2841-2857 (2010)). FUS(R495X) spontaneously co- condensed with G3BP1. However, when G3BP1-EGFP was sequestered by nGFP-NI, FUS(R495X) failed to form granules independently, indicating that G3BP1 contributes to the formation of FUS(R495X) inclusions (new Supplementary Fig. 11c,d). Collectively, these findings illuminate the biological impact of modulating the material properties of disease-relevant phase-separating proteins. We believe these additions have demonstrated the broad applicability of the STOP tool. For clarity, the above results have been included as Supplementary Fig. 11 in the revised manuscript.\nSupplementary Fig. 11 | a,b, Cell viability assessment of HeLa and U2OS cells coexpressing nGFP or nGFP-NI with G3BP1-EGFP under 500 µM arsenite stress for 2 h (a) or EGFP-TDP-43 NLSmut (b).\n8\nc,d, Representative images (c) and quantification (d) of condensates formation in HeLa cells coexpressing nGFP or nGFP-NI with FUS(R495X) and G3BP1-EGFP. Data in d was represented as mean ± s.d. of n = 3 biologically independent samples with at least 30 co-transfected cells per group.\nP values were determined using unpaired two-tailed Student’s t-tests (a,b), Welch’s one-way ANOVA followed by Games–Howell multiple comparisons test (d), All nGFP-fused proteins were detected using an anti-HA antibody (magenta). Scale bar, 20 μm (c).\nComment 2: 2. The authors propose STOP as a general tool for targeting SG components, based on its suppression of condensation in proteins such as CAPRIN1, FMR1, and PABPC1. However, it is unclear whether these effects arise from direct interactions with each protein or from indirect network-level perturbation via G3BP1 inhibition. Additionally, potential off-target effects on other membraneless organelles (e.g., P-bodies, nucleoli, nuclear speckles) are not assessed. Testing STOP in systems devoid of G3BP1, performing direct binding or co-localization assays, and evaluating its effects on unrelated condensates would help clarify its specificity and broaden the scope of its applicability.\nResponse 2: We thank the Reviewer’s suggestions. To address the reviewer's questions regarding the specificity of nGFP-NI's suppressive effect on other stress granule components, we conducted additional experiments in systems lacking G3BP1. These confirm that suppression results from direct interaction between nGFP-NI and the target protein, rather than indirect network-level effects.\nFirst, we expressed and purified EGFP-tagged CAPRIN1 and PABPC1 from E. coli for in vitro droplet formation assays. This in vitro system contained only Cy5-labeled nGFP-NI and the EGFP-fused target protein, excluding other cellular components such as G3BP1. Consistent with the G3BP1 results in Fig. 3e–f, pre-incubating nGFP-NI with either CAPRIN1 or PABPC1 prior to LLPS induction dramatically reduced droplet formation (new Supplementary Fig. 10d-f). These findings directly demonstrate that nGFP-NI acts through direct binding to the target protein.\nFurthermore, we expressed EGFP-FMR1 or EGFP-FXR2 in U2OS G3BP1/2 dKO cells. Both proteins formed cytoplasmic granules, and this granule formation was substantially reduced upon co-expression with nGFP-NI (new Supplementary Fig. 10g,h). These cellular results align with the in vitro data, reinforcing the conclusion that nGFP-NI-mediated suppression is direct and does not require the G3BP1-centered network. These results are included as Supplementary Fig. 10d-h in the revised manuscript.\nTo address the reviewer's concern regarding potential off-target effects, we monitored the morphology of other membraneless organelles—specifically P-bodies and nuclear speckles—during nGFP-NI targeting of G3BP1-EGFP under arsenite stress. No significant morphological changes were observed in these condensates (new Supplementary Fig. 4c–f). Furthermore, the P-body marker DCP1A showed no colocalization with G3BP1 condensates, and nuclear speckles reside within the nucleus, physically separated from cytoplasmic G3BP1 granules. These results indicate that nGFP-NI targeting of G3BP1-EGFP does not produce off-target effects on other 10 membraneless organelles. We further emphasize that nGFP-NI is only functional when bound to an EGFP-tagged target protein (new Supplementary Fig. 2f). Thus, it does not affect untagged proteins such as endogenous G3BP1, and the NI fragment alone is insufficient to perturb condensates without induced proximity (Supplementary Fig. 2b- e). The high selectivity and affinity of the nGFP–EGFP interaction further ensure the specificity of this tool. These data have been included as Supplementary Fig. 4c–f and Supplementary Fig. 2e,f in the revised manuscript.\nSupplementary Fig. 4 | c-f, Representative images of NaAsO -treated HeLa cells co-transfected with 2 G3BP1-EGFP and nGFP or nGFP-NI. Cells were treated with 500 µM NaAsO for the indicated time 2 (0, 0.5 h). The P-body marker DCP1A (c,d) or the nuclear speckle marker SC35 (e,f) were stained with their antibodies (red). Insets, regions of the indicated panels magnified 10 times. Right, fluorescence intensity profiles of G3BP1-EGFP (EGFP, green), nGFP or nGFP-NI (HA, magenta), and DCP1A (red) along the indicated line in the magnified images were plotted. Scale bars, 20 μm (c-f).\nComment 3: 3. The majority of the experiments rely on overexpression of tagged G3BP1 constructs (e.g., G3BP1-EGFP or Halo-tagged variants), which may not reflect physiological stoichiometry or native dynamics. This raises the possibility of artifacts in LLPS behavior or STOP-induced suppression. The efficacy of STOP in modulating SGs under endogenous protein levels remains unclear. The authors are encouraged to test STOP function in CRISPR knock-in cell lines expressing endogenous tagged G3BP1, or via complementation assays in G3BP1/2 knockout cells. Quantitative comparison of STOP versus endogenous protein expression would also be informative.\nResponse 3: We understand the Reviewer’s concern and thank the suggestions.\nTo address this concern, we used two well-established cell lines for stress granule research: U2OS G3BP1/2 dKO cells and U2OS cells stably expressing G3BP1-EGFP, which are widely acceptable and used for many studies on SGs (Gitler, A.D. et al. J. Neurosci. 34, 8083-97 (2014); Gwon, Y. et al. Science 372, eabf6548 (2021); Yang, P. et al. Cell 181, 325-345 (2020).; Zhang, K. et al. Cell 173, 958-971 (2018)). Notably, the stable U2OS G3BP1-EGFP line expresses the fusion protein at levels lower than endogenous G3BP1, minimizing potential overexpression artifacts. When we expressed 12 nGFP or nGFP-NI in this cell line and induced stress with arsenite, nGFP-NI suppressed SG formation as effectively as in HeLa cells overexpressing G3BP1-EGFP (new Supplementary Fig. 3b). Similarly, co-expression of G3BP1-EGFP with nGFP or nGFP- NI in U2OS G3BP1/2 dKO cells also showed clear SG suppression upon arsenite stress (new Supplementary Fig. 3c). These data are now included as Supplementary Fig. 3b,c.\nAdditionally, as demonstrated in Fig. 3a–d, FKBP-NI suppressed FRB-tagged G3BP1 granules in a rapamycin concentration-dependent manner. Because rapamycin concentration determines the effective amount of FKBP-NI bound to G3BP1-FRB, we propose that the overall suppression efficacy depends on the relative molar ratio of the G3BP1 module to its NI-binding partner, rather than on absolute protein levels.\nSupplementary Fig. 3 | b,c, Representative images and quantification of SGs in NaAsO -treated 2 U2OS G3BP1-EGFP stable cell line expressing nGFP or nGFP-NI (b), and the U2OS G3BP1/2 dKO cell line (c) coexpressing nGFP or nGFP-NI with G3BP1-EGFP. Cells were stressed with 500 µM NaAsO for 30 min (b) or 1 h (c). Data in b,c are represented as mean ± s.d. of n = 3 biologically 2 independent samples with at least 30 co-transfected cells per group. P values were determined using unpaired two-tailed Student’s t-tests. Scale bars, 20 μm (b,c).\nThe manuscript by Wang et al. proposes a previously uncharacterized non-catalytic function of human OGT where a fragment covering the N-terminal catalytic lobe and intervening domain (NI) of OGT appears to delay stress granule (SG) formation under sodium arsenite-induced stress. Based on this, the authors developed a modular tool that induces proximity of the OGT-NI domain to G3BP1 and other SG-nucleating proteins sharing similar domain architectures, thereby modulating SG formation. Mechanistically, the authors suggest that this modulation is achieved by altering the material properties of SGs, promoting more solid-like aggregates rather than liquid-like condensates.\nWhile this study potentially identifies a novel (non-catalytic role) of OGT in modulating stress granule formation and introduces a promixity-inducing approach (albeit relying on existing tools/approaches) there are significants flaws that make the solidity of the conclusions drawn questionnable and therefore this is not a reliable advance of scientific knowledge.\nResponse: We thank the reviewer for their positive recognition of our novel findings and for their thoughtful critiques, which have greatly strengthened our study. In response to the reviewer's constructive suggestions, we have performed additional experiments and revised the manuscript accordingly.\nTo fully address the reviewer’s concerns, like our responses to Reviewer #2, we have incorporated the following new evidence, as outlined below:\n1) To address the concern regarding protein overexpression and cell lines, we included additional validation experiments in U2OS cells stably expressing G3BP1-EGFP, and U2OS G3BP1/2 dKO cells.\n2) To better demonstrate the biological relevance of nGFP-NI-induced SG suppression, we added data on its effects on cell viability and its ability to perturb condensates formed by disease-associated proteins.\n3) To elucidate the mechanism of nGFP-NI action, we incorporated new data from cross- linking mass spectrometry, aggregate separation assays, and confocal imaging.\nWe also thank the reviewer for pointing out several minor errors, all of which have been corrected in the revised manuscript for improved clarity and accuracy. Our detailed point-by-point responses follow below.\nMajor concerns\nComment 1: 1) Physiological relevance. The findings rely heavily on 1) overexpression, and\nAs detailed in our Response 3 to Reviewer #2 Comment 3, we validated the effect of nGFP-NI in U2OS cells stably expressing G3BP1-EGFP and in U2OS G3BP1/2 dKO cells rescued with G3BP1-EGFP. The results from both cell lines were consistent with our previous observations in HeLa cells (new Supplementary Fig. 3b,c). These cell lines are well-established models in SG research (Gitler, A.D. et al. J. Neurosci. 34, 8083-97 (2014); Gwon, Y. et al. Science 372, eabf6548 (2021); Yang, P. et al. Cell 181, 325-345 (2020); Zhang, K. et al. Cell 173, 958-971 (2018)). Importantly, the U2OS cell line stably expressing G3BP1-EGFP maintains the fusion protein at levels lower than endogenous G3BP1, thereby minimizing potential artifacts associated with overexpression.\nSupplementary Fig. 3 | b,c, Representative images and quantification of SGs in NaAsO -treated 2 U2OS G3BP1-EGFP stable cell line expressing nGFP or nGFP-NI (b), and the U2OS G3BP1/2 dKO cell line (c) coexpressing nGFP or nGFP-NI with G3BP1-EGFP. Cells were stressed with 500 µM NaAsO for 30 min (b) or 1 h (c). Data in b,c are represented as mean ± s.d. of n = 3 biologically 2 independent samples with at least 30 co-transfected cells per group. P values were determined using unpaired two-tailed Student’s t-tests. Scale bars, 20 μm (b,c).\n2) a system that forces OGT to the stress granules where (even according to the authors’ own experiments) it does not occur/belong\n3) the use of a carcinogenic cell line (HeLa) with questionnable physiological relevance.\nCellular stress is a universal phenomenon affecting virtually all cell types, which explains why stress granule research spans diverse biological systems. Although SGs are frequently studied in the context of neurodegenerative diseases, they also play critical roles in other pathological conditions such as cancer, viral infection, and cardiovascular disease (Gao, X. et al. Br. J. Pharmacol. 176, 4421-4433 (2019); Grabocka, E. & Bar- Sagi, D. Cell 167, 1803-1813 (2016); Jayabalan, A.K., Griffin, D.E. & Leung, A.K.L. Viruses 15, 449 (2023); Panas, M.D. et al. PLoS Pathog. 11, e1004659 (2015); Yang, G. et al. Cardiovasc. Transl. Res. 18, 738-748 (2025)). Notably, growing evidence indicates that SGs not only influence the balance between cell survival and death but also contribute to cancer invasion, metastasis, and drug resistance (Lavalee, M. et al. Trends Cancer 7, 902-915 (2021); Khong, A. et al. bioRxiv (2022)). Established cell models including HEK293T, HeLa, and U2OS have been widely used in this field for decades, as evidenced by extensive work from leading laboratories such as those of Clifford P. Brangwynne, J. Paul Taylor, Roy Parker, and Anthony A. Hyman.\nWe thank the reviewer for emphasizing physiological relevance. In response, and as detailed in our Response 1 to Reviewer #2 Comment 1, we conducted additional experiments to further strengthen the biological relevance of our findings beyond those presented in Fig. 6g–i. These include assessing cell viability following targeting of G3BP1 and a disease-associated TDP-43 mutant (new Supplementary Fig. 11a,b) and using the tool to elucidate the co-condensation behavior of the FUS(R495X) mutant with G3BP1 (new Supplementary Fig. 11c,d).\nc,d, Representative images (c) and quantification (d) of condensates formation in HeLa cells coexpressing nGFP or nGFP-NI with FUS(R495X) and G3BP1-EGFP. Data in d was represented as mean ± s.d. of n = 3 biologically independent samples with at least 30 co-transfected cells per group.\nP values were determined using unpaired two-tailed Student’s t-tests (a,b), Welch’s one-way ANOVA followed by Games–Howell multiple comparisons test (d), All nGFP-fused proteins were detected using an anti-HA antibody (magenta). Scale bar, 20 μm (c).\nIt remains unclear whether the OGT-NI domain has a physiological role in SG modulation under endogenous expression levels. Does the NI domain delay SG formation without overexpression? What happens in a stable cell line carrying point mutations or deletion in the specific OGT domains?\nWe thank the reviewer for these questions. The OGT-NI fragment itself has no discernible effect on SGs (Supplementary Fig. 2b-e). SG modulation by OGT-NI occurs only when coupled with an induced-proximity system, such as the nGFP–GFP pair (Fig. 2), the rapamycin-dependent FKBP–FRB pair (Fig. 3a,c), or the AP1867-Cl-induced FKBPF36V–HaloTag pair (Fig. 1a,c). Thus, the NI domain alone does not perturb SG formation at any expression level. Correspondingly, nGFP-NI did not delay endogenous SG formation in the absence of its EGFP-tagged G3BP1 target (Supplementary Fig. 2f).\nMeanwhile, the induced proximity modules ensure the selectivity of this nGFP-NI tool, minimizing potential off-target effects.\nAs the Reviewer suggested, we generated three stable HeLa cell lines expressing the inactive nGFP-OGT(K852A), nGFP-∆NI and NI alone, respectively, using lentivirus infection. We examined the SG formation in all three cell lines. As expected, nGFP- OGT(K852A) retained the ability to suppress SGs; nGFP-∆NI failed to perturb SGs due to the absence of the functional NI fragment; and NI alone, lacking the nGFP proximity module, was also ineffective (new Supplementary Fig. 3a). These results align with our previous data in Fig. 2d,e and Supplementary Fig. 2b, confirming that both the induced- proximity module (e.g., nGFP) and the NI fragment are essential for SG suppression.\nThese data are now presented in Supplementary Fig. 3a.\nSupplementary Fig. 3 | a, Representative images of HeLa cells stably expressing nGFP- OGT4(K852A), nGFP-∆NI (a truncation lacking the N-cat and Int-D domains), or the NI fragment alone when transfected with empty vector (control) or G3BP1-EGFP. After exposure to NaAsO stress, cells 2\n18\nwere fixed and stained with anti-HA (magenta, detect the nGFP-fusion proteins) and anti-G3BP1 (red) antibodies. Scale bars, 20 μm.\nComment 2: 2) Producing an isolated ND-NI fragment is a blunt tool. The N-terminal and intervening domains of OGT are not at all “domains”. They are closely packed forming a continuous catalytic domain. Chopping them up like this will exposed a hydrophobic core, producing insoluble protein, likely to precipitate, for instance, in/on stress granules. Nowhere do the authors show that this is a bona fide soluble protein in an Eppendorf tube when produced in vitro.\nResponse 2: We adopted the domain annotations from a well-established OGT structural study by Suzanne Walker lab (Lazarus, M.B. et al. Nature 469, 564-567 (2011)). In that paper, the authors said that “… The catalytic region contains three domains: the amino (N)-terminal domain (N-Cat), the carboxy (C)-terminal domain (C-Cat), and the intervening domain (Int-D) (Fig. 2d)…”\nWe agree with the reviewer that truncating OGT could alter its physicochemical properties, including solubility. Consistent with this, we found that while a portion of nGFP- NI remained soluble when expressed in E. coli, some was retained in inclusion bodies. Ultimately, we successfully purified soluble nGFP-NI at a concentration of 0.34 mg/mL in PBS buffer (300 mM NaCl, 10% glycerol, pH 7.4), confirming its solubility in vitro.\nFurthermore, as shown in Fig. 3a, overexpressed FKBP-NI does not localize to G3BP1 granules without rapamycin induction, indicating that it is unlikely to precipitate within SGs. During revision, we also expressed nGFP-NI in HeLa cells and monitored its distribution during a 2-hour arsenite treatment. While endogenous G3BP1 formed stress granules normally after 0.5 hours, nGFP-NI remained diffusely cytoplasmic without forming puncta (new Supplementary Fig. 2f). This suggests that nGFP-NI does not precipitate into SGs under these conditions.\nFurthermore, as described in our Response 1 to Reviewer #2 Comment 1, we separated the RIPA-soluble and insoluble fractions under the same experimental conditions as used in new Supplementary Fig. 2f. It is important to note that a majority of both nGFP-OGT4 and nGFP-NI remained soluble under these conditions.\nEndogenous G3BP1 remained in the RIPA-soluble fraction even after arsenite-triggered LLPS. Notably, nGFP-NI began to appear in the insoluble fraction after just 0.5 hours of stress, indicating a propensity for aggregation. nGFP-OGT4 also transitioned to the insoluble fraction, albeit more slowly. These data suggest that nGFP-OGT4/NI tend to aggregate in response to arsenite stress.\nIn addition, we would like to emphasize that apart from nGFP-NI, multiple OGT fusions, such as OGT-FKBPF36V (full-length OGT, Fig. 1a), full-length OGT-His (Fig. 1e, g), nGFP-OGT4 (Fig. 2b-e, Supplementary Fig. 3a), are all able to suppress G3BP1 condensates to varying degrees. The nGFP-OGT4 construct is adopted from previous publications: the human OGT crystal structure (hOGT4.5) from Suzanne Walker lab (Lazarus, M.B. et al. Nature 469, 564-567 (2011)) and a targeted protein O-GlcNAcylation tool from Christina Woo’s lab (Ramirez, D.H. et al. ACS Chem. Biol. 15, 1059–1066 (2020), addgene: Plasmid #160739). In all these instances, the expressed OGT proteins have been reliably confirmed to be soluble.\nComment 3: 3) Biological Utility of the Tool. Although the tool delays SG formation, it also promotes solid-like G3BP1 aggregation. How beneficial is this in the broader context of SG biology? Would this perturbation itself have deleterious consequences for cell physiology?\nWe agree with the reviewer on the importance of understanding the biological consequences of this phase transition. Although stress granules are often studied in the context of neurodegenerative diseases, they also play critical roles in other pathological conditions such as cancer, viral infection, and cardiovascular disease (Gao, X. et al. Br. J. Pharmacol. 176, 4421-4433 (2019); Grabocka, E. & Bar-Sagi, D. Cell 167, 1803-1813 (2016); Jayabalan, A.K., Griffin, D.E. & Leung, A.K.L. Viruses 15, 449 (2023); Panas, M.D. et al. PLoS Pathog. 11, e1004659 (2015); Yang, G. et al. Cardiovasc. Transl. Res. 18, 738-748 (2025)). In our previous manuscript version, we demonstrated how nGFP-NI- induced disruption of G3BP1 LLPS affects nucleocytoplasmic transport (Fig. 6g,h) and cell viability (Fig. 6i), highlighting the tool's potential for perturbing SG function. During revision, we further investigated whether nGFP-NI–mediated targeting of G3BP1 and G3BP1 influences granules containing disease-associated proteins. Perturbation of granules formed by a disease-related TDP-43 mutant with nGFP-NI similarly reduced cell viability, suggesting that TDP-43 solidification accelerates cell death under stress (new Supplementary Fig. 11a,b). These results reinforce the established concept that disrupting normal liquid-like SG dynamics can impair viability—as reported in other studies (Johnson, B.S. et al. J. Biol. Chem. 284, 20329-20339 (2009); Protter DSW, Parker R. Trends. Cell Biol. 26, 668-679 (2016))—and underscore the general applicability of nGFP-NI, as further supported by Fig. 6a–f.\nIn addition, we co-transfected the disease-associated mutant FUS(R495X) together with G3BP1-EGFP and nGFP or nGFP-NI into HeLa cells according to previous studies (Dormann, D. et al. EMBO J. 29, 2841-2857 (2010)). FUS(R495X) spontaneously co- condensed with G3BP1. However, when G3BP1-EGFP was sequestered by nGFP-NI, FUS(R495X) failed to form granules independently, indicating that G3BP1 contributes to the formation of FUS(R495X) inclusions (new Supplementary Fig. 11c,d). Collectively, these findings illuminate the biological impact of modulating the material properties of disease-relevant phase-separating proteins. We believe these additions have demonstrated the broad applicability of the STOP tool. For clarity, the above results have been included as Supplementary Fig. 11 in the revised manuscript.\nc,d, Representative images (c) and quantification (d) of condensates formation in HeLa cells coexpressing nGFP or nGFP-NI with FUS(R495X) and G3BP1-EGFP. Data in d was represented as mean ± s.d. of n = 3 biologically independent samples with at least 30 co-transfected cells per group.\nP values were determined using unpaired two-tailed Student’s t-tests (a,b), Welch’s one-way ANOVA followed by Games–Howell multiple comparisons test (d), All nGFP-fused proteins were detected using an anti-HA antibody (magenta). Scale bar, 20 μm (c).\nComment 4: 4) Context-Specificity of Discovery. The role of OGT-NI in SG regulation appears limited to G3BP1 or G3BP1-like proteins under arsenite stress. However, other SG-forming proteins (e.g., UBAP2L, eIF4G1) contribute to SG assembly under different stress conditions (e.g., osmotic, ER stress). The extent to which this mechanism generalizes remains unclear.\nResponse 4: We apologize for any lack of clarity in our previous manuscript version regarding the general applicability of the mechanism. We would like to take this opportunity to clarify the generality of this tool.\nFirst, we would like to define the specificity of this tool. Although most experiments were conducted under arsenite stress, our results indicate that nGFP-NI modulates the liquid-liquid phase separation (LLPS) of target proteins by directly altering their material 23 properties, rather than by interfering with upstream stress granule pathways (Supplementary Fig. 5). We discussed this point in the main text from line 203 to line 207: “These data suggest that early steps prior to G3BP1 involvement, such as stress recognition and translational arrest, proceeded normally, which in turn supports the notion that the inhibitory effect of nGFP-NI on SG formation does not stem from interference with the upstream SG signaling pathways, but likely from direct perturbation on G3BP1 itself.” This conclusion is further supported by in vitro droplet formation assays, as detailed in Response 2 to Reviewer #2 Comment 2 and shown in new Supplementary Fig. 10e,f.\nTherefore, nGFP-NI functions as a protein-specific tool for condensate regulation under stress, not merely as a context-specific agent limited to arsenite-induced SGs.\nThis conclusion can be further supported by Fig. 2g-h, which are discussed in the main text from line 182 to line 190 as follows: “However, it is important to note that G3BP1 may not exhibit the same degree of centrality under other stress conditions. To determine whether nGFP-NI is applicable to SG suppression under different stressors, we tested its effect on G3BP1-EGFP in HeLa cells subjected to osmotic stress (sorbitol) or ER stress (DTT). As expected, we observed a significant reduction in G3BP1 granules (Fig. 2g,h). However, unlike the arsenite treatment, other SG markers such as UBAP2L and eIF4G1 formed SGs independent of G3BP1 under sorbitol-induced stress (Supplementary Fig. 4b). These results suggest that nGFP-NI effectively suppresses G3BP1-dependent SGs and provides a strategy for distinguishing the relative importance and centrality of G3BP1 under different stress conditions.” These results suggest that nGFP-NI is a protein-specific tool no matter of the treatments of which LLPS triggers.\nFurthermore, as shown in Fig. 5, the suppressive effect of nGFP-NI depends on recognizing the domain architecture of the target protein rather than its precise primary sequence. Therefore, we selected CAPRIN1, PABPC1, FMR1, and FXR2 to demonstrate generality because they share a similar domain organization with G3BP1. Notably, FMR1 and FXR2 form granules even in the absence of stress. In contrast, eIF4G1 and UBAP2L—which possess distinct architectures—are unaffected by nGFP-NI (Supplementary Fig. 10i,j). The generality we emphasize is the ability to regulate LLPS across multiple proteins, particularly several SG components. We propose that this approach may be extendable to other phase-separating proteins with analogous domain architectures, potentially enabling modulation of condensates beyond SGs. A broader discussion of this generality can be found in the Discussion section.\nSpecific comments:\nResponse: We thank the Reviewer for pointing out some mistakes and confusion. We have now corrected all of them and provide additional clarifications.\nResponse 5: The final construct, nGFP-NI, does contain the N-catalytic domain. However, it remains completely devoid of enzymatic activity. According to the OGT structure (Lazarus, M.B. et al. Nature 469, 564-567 (2011)), the N-catalytic and C-catalytic domains must come together to form a complete catalytic pocket. The substrate UDP-GlcNAc binds in a pocket within the C-cat domain near the interface with the N-cat domain. Therefore, since nGFP-NI includes only the N-cat domain and the Int-D, it cannot possess any catalytic activity.\nMoreover, we have included many experiments and controls to demonstrate that this effect is independent of OGT’s catalytic activity. First, we used OGT K852A as an inactive control in many experiments. Mutation of the OGT K852 site is a golden standard for generating inactive OGT. Functions revealed by this mutant are recognized as non- catalytic (Levine, Z.G. et al. Proc. Natl. Acad. Sci. USA 118, e2016778118 (2021)). Second, in all the in vitro droplet formation assays performed in this study, the experimental conditions preclude any possible glycosylation reactions, as no UDP- GlcNAc is present. Third, Int-D alone, when fused to nGFP (Fig. 2d,e), still affects G3BP1 LLPS, even though it does not contain any catalytic domains. Collectively, these data confirm that the effect of this tool is independent of OGT’s catalytic activity.\nComment 6: The interaction between OGT and G3BP1 is strongly dependent on the affinity between nGFP and EGFP. Is this mechanism even possible in vivo or is it a forced interaction?\nResponse 6: Our design of the nGFP-NI tool originated from the unexpected finding that OGT can suppress SGs (or G3BP1 condensates) upon induced proximity. This encouraged us to engineer OGT into a compact and selective tool for SG modulation, represented by nGFP-NI. We have demonstrated that the suppression effect depends on the induced proximity between OGT-NI and the target protein, such as G3BP1 (Fig. 3a,c, Supplementary Fig. 6c,d).\nNotably, a key highlight of this nGFP-NI tool is its modularity: we can replace the nGFP–GFP pair with any other chemically or light-induced dimerization system for improved spatiotemporal and concentration-dependent control over condensates. We have already demonstrated this modularity by presenting data based on the rapamycin- facilitated FKBP–FRB pair and the AP1867-induced FKBPF36V–HaloTag pair. The nGFP– 25 GFP pair could also be replaced with natural ligands, such as nanobodies against endogenous proteins or natural binding peptides, which may facilitate future in vivo applications. Many studies have shown the feasibility of induced-proximity tools in vivo (Zhang, Y. et al. Nature 643, 1107-1116 (2025)), and we have included this vision in the Discussion section.\nComment 7: Figure 1: 60% normal SG formation vs 40% reduced SG. The authors should comment on that.\nResponse 7: We suspect that the Reviewer may be referring to the OGTAC-2 treatment group in Fig. 1d. In HeLa cells treated with OGTAC-2 and arsenite, nearly 60% of cells were SG-positive, suggesting that the remaining 40% showed no obvious SGs. This experiment was conducted in CRISPR knock-in FKBPF36V-OGT HeLa cells overexpressing G3BP1-Halo. Heterogeneity in G3BP1-Halo transfection and expression among individual cells led to a phenomenon wherein, in some cells, endogenous OGT levels were sufficiently high to suppress G3BP1, while in others, G3BP1 levels exceeded those of OGT and could not be effectively suppressed following the addition of OGTAC- 2.\nComment 8: Extended Fig. 1b:\n1. Why is there a FLAG tag band in lane 1, despite no protein being expressed?\n2. Why are HIS-tag bands present in lanes 1 and 8?\n3. Why are there no OGT bands in lanes 5–8 and 12–13, where OGT(K842A) was used? Why did they use this specific mutation (the rest of the field tends to use the isosteric K842M mutation)?\n4. Why does the HIS tag disappear in lane 4?\nResponse 8: Thank you for pointing out these mistakes. We have replaced them with new figures (new Supplementary Fig. 1b,d).\nWe suspected that the disappearance of protein bands was caused by degradation. Therefore, we repeated the experiment using freshly prepared purified proteins and total RNA, and successfully observed all expected bands (Fig. R1). Because the original data were intended to demonstrate that G3BP1 cannot be glycosylated by OGT in vitro, we replaced the figure with a new data to avoid potential confusion. The new figure shows the results of in vitro glycosylation assays on His-tagged G3BP1-EGFP and EGFP- YTHDF3 (new Supplementary Fig. 1d).\nSupplementary Fig. 1 | d, Immunoblots of G3BP1-EGFP and EGFP–YTHDF3 and their O-GlcNAc levels following an in vitro O-GlcNAcylation reaction. RL2, anti-O-GlcNAc antibody.\nWe used OGT(K852A) as the inactive control and would like to clarify two points.\nFirst, the K852 site is identical to the “K842” site mentioned in Suzanne Walker’s Nature paper (Lazarus, M.B. et al. Nature 469, 564-567 (2011)). Our numbering follows the UniProt entry (https://www.uniprot.org/uniprotkb/O15294/entry). Both K852A and K852M mutants have been widely used in the field as inactive controls. For example, both mutants have been used in publications from the Suzanne Walker lab, which has conducted pioneering work on OGT structure, function, and biochemistry (Lazarus, M.B. et al. Science 342, 1235-1239 (2013); Levine, Z.G. et al. Proc. Natl. Acad. Sci. USA 118, e2016778118 (2021)). The nGFP-OGT4(K852A) construct was a generous gift from the Christina Woo lab (Ramirez, D.H. et al. ACS Chem. Biol. 15, 1059–1066 (2020); Addgene: Plasmid #160741).\nComment 9: 5. Extended Fig. 1b vs. 1c: These panels seem contradictory. In 1b, OGT does not appear to interact with G3BP1, whereas in 1c, OGT co-immunoprecipitates with G3BP1. Please clarify this discrepancy.\nResponse 9: Thank you for pointing this out. The experiments in Extended Fig. 1b (now Supplementary Fig. 1b) were performed in CRISPR knock-in FKBPF36V-OGT HeLa cells overexpressing G3BP1-Halo, where OGT is present at relatively low endogenous levels. Therefore, the interaction between OGT and G3BP1 could only be prominently detected 28 after the addition of OGTAC-2 to induce proximity. In Extended Fig. 1c (now Supplementary Fig. 1c), both OGT and G3BP1-EGFP were overexpressed, allowing weak interactions between OGT and G3BP1 to be observed.\nComment 10: Figure 2b: What is the sequence of the OGA(stalk) construct used?\nResponse 10: The nGFP-OGA(stalk) construct used in Fig. 2 is from our previous publication (Ge, Y. et al. Nat. Chem. Biol. 17, 593-600 (2021); Addgene: Plasmid #168097). For clarity, we have summarized all plasmids used in this study in Supplementary Table 1 and annotated their Addgene information for reference.\nComment 11: Extended Fig. 2a / Line 158-162: The HA-nGFP-NI construct is used in Fig. 2a before it is introduced in the main text (line 162). Please reorder to avoid confusion.\nResponse 11: Thank you for your suggestion, and we have revised the text accordingly.\nComment 12: Extended Fig. 2e: The co-localization of G3BP1 with eIF4G1 under sorbitol-induced stress appears different from UBAP2L. Please comment on this observation.\nResponse 12: In Extended Fig. 2e (now Supplementary Fig. 4b), the co-localization of G3BP1 with eIF4G1 is comparable to its co-localization with UBAP2L. The intensity profiles show that peaks from both UBAP2L and eIF4G1 signals align with those of G3BP1-EGFP (as indicated by the overlapping green and red peaks), indicating strong co-localization. The height of the peaks reflects the fluorescent intensity in each channel, which may vary due to differences in antibody efficiency and other experimental conditions. Upon re-examination, we identified an error in the analysis of fluorescence intensities. We apologize for careless errors and now correct this figure as shown below.\nComment 13: Figure 3e: The interaction of the truncated versions of OGT is forced with the nanobody/GFP. What happens when the interaction is not forced (like in Figure 1g)?\nResponse 13: As our Response 1 to Reviewer #3 Comment 1, induced proximity is essential for suppressing G3BP1 LLPS in cells, a conclusion supported by data in Fig. 3a-d, Supplementary Fig. 2b-f.\nFig. 1g presents an in vitro droplet formation assay. Although purified wild-type OGT was used in this experiment, total RNA was added to promote G3BP1 phase separation— a process that can also be viewed as a protein concentrating or crowding mechanism. Furthermore, both G3BP1 and OGT were present at high concentrations (25 µM and 5– 10 µM, respectively). Compared to the truncated OGT, wide-type OGT still contains the TPR domain for protein interaction (e.g. G3BP1). Under these conditions, weak interactions between OGT and G3BP1 can also take effect. In conclusion, interaction between OGT variants and G3BP1 is required for the SG suppressive effect.\nComment 14: Extended Fig. 3: The figure labelling does not match the legend. Please revise for consistency.\nResponse 14: Thank you for pointing it out. We have corrected it.\nComment 15: Extended Fig. 3b: At 0 µM rapamycin, HA signal is observed in FLAG pull- down, suggesting baseline interaction. Does this impact the interpretation of the results?\nResponse 15: Thank you for pointing this out. We have replaced it with a new figure after immunoprecipitation protocol optimization (new Supplementary Fig. 6b), where no HA signal was observed in FLAG IP at 0 nM rapamycin. Even if a baseline interaction were present possibly due to protein overexpression, the amount of interacting protein would likely be insufficient to produce a functional effect. As shown in Fig. 3b, a reduction in SGs is observable with rapamycin concentrations as high as 100 nM.\nComment 16: Data analysis of some data (like granule area) shouldn't have been done with a parametric test, since data is not normally distributed.\nResponse 16: Thank you for highlighting the non-normality of the granule area data. As the Reviewer suggested, we therefore replaced all parametric tests with their non- parametric counterparts: for comparisons involving three or more groups (e.g., Fig. 1h), we adopted the Kruskal–Wallis test followed by Dunn’s multiple comparisons test; for two- group comparisons (e.g., Fig. 3e,f), we used two-tailed Mann–Whitney U tests. In addition, we have revised several other analyses, updated all corresponding p-values, and adjusted the figure legends throughout the revised manuscript.\nComment 17: Line 52: Please explain or reference what “NTF2L-targeting” refers to.\nResponse 17: We apologize for any confusion. The NTF2L domain refers to the N- terminal NTF2-like domain of G3BP1, which is responsible for its dimerization. The intended meaning is that viral peptide mimetics or small molecules are developed to target the NTF2L domain of G3BP1. We have revised the relevant sentences to improve clarity.\nComment 18: Line 53: Sentence lacks a reference.\nComment 19: Line 55: Why can’t existing regulatory tools for G3BP1/2 be adapted to other SG proteins?\nResponse 19: This is because existing regulatory tools are small molecules or peptides that were selectively designed or screened to target the NTF2L domain of G3BP1/2. Since every stress granule protein has distinct sequences and structures, these molecules cannot be applied to other proteins. We have also revised the original sentence to prevent any potential confusion.\nComment 20: Line 97: “We synthesized and applied a chimeric molecule (…)” The phrasing suggest that the molecule is a new creation – this is not the case. Rephrasing as “resynthesized” would be more precise.\nResponse 20: Thank you for this suggestion. We have corrected it to \"resynthesized.\" Furthermore, to improve clarity, we have replaced the small molecule name \"AP1867-Cl\" with \"OGTAC-2,\" the designation used in its original publication (Ma, B. et al. J. Am. Chem. Soc. 146, 9779–9789 (2024)), throughout the manuscript.\nComment 21: Line 186: “Rapamycin treatment without inducing observable cellular stress”. Please elaborate. What indicators were assessed to conclude that there was no observable stress?\nResponse 21: As shown in Supplementary Fig. 6a, we did not observe any SGs even with G3BP1-FRB and FKBP-NI overexpression and the treatment of Rapamycin as high as 1000 nM; therefore, we concluded that \"rapamycin treatment without inducing observable cellular stress.\" For clarity, we have now revised this sentence as below:\n“…rapidly forming a ternary complex as its concentration increases without inducing detectable SGs…”\nComment 22: Line 210: The text suggests that the phenotype “may not stem from disruption of G3BP1’s RNA-binding capacity.” Please provide more evidence or rationale to support this claim.\nResponse 22: We observed that nGFP-NI–mediated suppression of G3BP1 LLPS remains effective even when PEG8000 is used as the LLPS trigger (Fig. 3f). Since PEG8000-induced G3BP1 LLPS does not rely on RNA binding, these results allow us to exclude the hypothesis that nGFP-NI suppresses G3BP1 LLPS by preventing its interaction with RNA. To avoid confusion, we have removed this claim from the current version of the manuscript.\nResponse 23: We apologize for these confusions. We have revised this part as below:\n“…These data suggest that early steps prior to G3BP1 involvement, such as stress recognition and translational arrest, proceeded normally,…” (line 203 to line 207). Also, in accordance with our Response 1 to Reviewer #2 Comment 1, we have now revised all the relevant mechanistic descriptions for clarity.\nComment 24: Line 231: The text refers to “sequestering TDP-43 together,” but TDP-43 is not observable in Extended Fig. 4c.\nResponse 24: We apologize for the lack of clarity in these images.\nTDP-43 does partition into G3BP1 granules. We regret that the TDP-43 signal was difficult to discern, likely due to limitations in resolution and fluorescent intensity in the original images. We have now re-processed and magnified the TDP-43 images, as shown below. These updated images have now been included as Supplementary Fig. 8a.\nComment 25: Line 233 / Fig. 4a: The statement claims that puncta in nGFP-NI-expressing cells are more aggregated, but Fig. 4a seems to show this phenotype in nGFP-only expressing cells. Please verify.\nResponse 25: We have enlarged the images originally presented in Fig. 4a. As shown below, after two hours of stress, SGs in control cells expressing nGFP-only retained a spherical morphology, indicative of liquid-like properties. In contrast, the puncta in cells expressing nGFP-NI appeared more aggregated and irregular, suggesting a solid-like state. These data are presented as a new Fig. 4a.\nComment 26: Line 252 / Extended Fig. 4j: The claim about increased eIF4G1 inclusion is not supported, as no eIF4G1 bands are visible in the figure.\nResponse 26: In Extended Fig. 4j (now Supplementary Fig. 9d), eIF4G1 bands are present. We have pointed these bands out below (Fig. R2):\nResults in Fig. 4a can also support this claim.\nReviewer #1 (Remarks to the Author):\nIn the revised manuscript, the authors have performed additional experiments such as the use of a RIPA lysis buffer and subsequent analysis of the soluble and insoluble fractions to address my previous concerns regarding the role of protein aggregation in this work. In light of the new results, the authors have proposed an updated dual-effect mechanism for the action of nGFP-NI, thereby significantly improving the impact of the findings reported herein. Additionally, the employment of cross-linking mass spectrometry and the live-dead assays have provided more insights into the structural and physiological implications of the SG modulation. Therefore, I recommend the manuscript for publication.\nResponse: We thank the Reviewer for supporting the manuscript for publication.\nReviewer #2 (Remarks to the Author):\nMy concerns have been addressed with additional experiments and explanation. I don't have further question and support publication of this work in NC.\nResponse: We thank the Reviewer for supporting the manuscript for publication.\nReviewer #4 (Remarks to the Author):\nThe manuscript under review is Wang et al., NCOMMS-25-44788A. Per [Redacted] , my remit was to assess whether the revised manuscript and author rebuttal adequately address Reviewer 3’s critique from the first round of review. I find that they do. The authors have done an admirable, comprehensive job of adding new data, revising the manuscript, and carefully answering the reviewer’s comments. Below are a few minor outstanding points that the authors should address through straightforward text revisions. After that, I believe Reviewer 3’s concerns will have been satisfied and the manuscript will likely be suitable for publication.\nResponse: We are grateful for the Reviewer 4's positive assessment of our revised manuscript and our responses to Reviewer 3’s comments.\nResponse 1: We thank the Reviewer for this suggestion and have added one sentence to describe the uncertainty as below:\n“…However, due to the overexpression of G3BP1-Halo, endogenous OGT may not be enough to target all G3BP1-Halo proteins in most cells, leading to insufficient SG suppression…” (line 104 to line 106).\nComment 2. Throughout the manuscript, the authors should ensure that the text does not imply any physiological function for full-length, endogenous OGT in suppressing SG formation because (as the authors acknowledge elsewhere) this has not been demonstrated. Some passages may still require revision. For example, the Abstract reads “Here, we identified OGT to suppress the LLPS of G3BP1, a key SG protein, and thereby SG assembly.” This makes it sound like the authors are claiming a physiological role for endogenous OGT in suppressing SG formation, when in fact their claim is that an artificial condition – induced proximity – is needed for OGT (or a fragment thereof) to exert these effects.\nResponse 2: We thank the Reviewer’s suggestion and have revised the text at several places to emphasize the induced-proximity–dependent function of OGT. These revisions are as below:\n1. Line 22: “…we identified OGT to suppress the LLPS of a key SG marker G3BP1 upon induced proximity and thereby…”\n2. Line 109: “…we speculate that OGT may be one of the factors exhibiting “negative cooperativity” for SG regulation when directly interacting with G3BP1.”\n3. Line 381: “…Here, we surprisingly found that OGT—long studied for its enzymatic activity—suppresses G3BP1-centric SGs upon induced proximity independent of O- GlcNAc installation (Fig.1).”\n4. Line 436: “…The ‘adaptor protein hypothesis’ posits that adaptor proteins may act like the induced proximity modules to help target OGT to selective cellular substrates for diverse functions28,59.”\nResponse 3: Thank you for this suggestion. We have now highlighted this future direction and acknowledged the limitation of the cell lines used in our study. The following text has been added to the Discussion:\n“Therefore, extending the application of STOP from current cultured cell lines to other models, such as primary or iPSC-derived neurons51, would be a critical next step to fully realize its potential.” (line 406 to 408)\nComment 4. I’m not clear on the authors’ vision of how STOP recognizes the “domain architectures” of G3BP1, CAPRIN1, PABPC1 and FMR1/FXR2. As the authors note, these proteins differ significantly in their primary sequences (and, as the authors do not note, in the spacing between the domains and in the structures of the domains), so they all look pretty different, despite sharing the general functional organization of oligomerization-IDR-RNA binding. Given this, I’m not sure how, exactly, the authors think STOP is able to bind to all these disparate proteins to inhibit SG formation. It would be helpful to have a few sentences or short paragraph in the Discussion that explains a testable hypothesis or model for how STOP recognizes these different SG proteins at the biochemical or structural level.\nResponse 4: We thank the Reviewer for this suggestion and apologize for the lack of clarity. We have now added a few sentences in the Discussion to explain our hypothesis. The revised text is shown below:\n“…Proteins essential for assembling diverse condensates, such as SG, P-body and Paraspeckle, primarily rely on stable oligomerization and RNA binding—rather than IDR- mediated weak interactions—to achieve high network connectivity for LLPS16. We speculate that targeted immobilization by STOP may impair their oligomerization and RNA-binding equilibria in the soluble phase, thereby shifting the LLPS threshold. This hypothesis could be tested by tuning STOP–target binding affinity or by comparing its effects on proteins whose LLPS is driven mainly by IDRs.” (line 412 to 418)\n3"}]

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