Stress Granule Assembly Research Articles

The non-canonical RNA binding protein RAN stabilizes the mRNA of intranuclear stress granule assembly factor G3BP1 in nasopharyngeal carcinoma

RNA binding proteins play critical roles in tumor progression by participating in the post-transcriptional regulation of RNA. However, the levels and function of RBPs in nasopharyngeal carcinoma (NPC) remain elusive. Here we identified a non-canonical RNA binding protein RAN that has the most significant role in NPC progression by a small siRNA pool screening. Functionally, RAN facilitates NPC proliferation and metastasis in vitro and in vivo. High levels of RAN are associated with poor prognosis of NPC patients and can be performed as a prognostic biomarker. Mechanistically, RAN increases the nucleus import of TDP43 and enhances TDP43 nuclear distribution. On the other hand, RAN is directly bound to the coding sequence of G3BP1 mRNA and serves as an adapter to facilitate TDP43 interacting with G3BP1 mRNA 3′ untranslated region. These contribute to increasing G3BP1 mRNA stability in the nucleus and lead to up-regulation of G3BP1, which further enhances AKT and ERK signaling and ultimately promotes NPC proliferation and metastasis. These findings reveal that RAN stabilizes intranuclear G3BP1 mRNA by dual mechanisms: recruiting TDP43 into the nucleus and enhancing its interaction with G3BP1 mRNA, suggesting a critical role of RAN in NPC progression and providing a new regulation framework of RBP-RNA.

Stress Granule Assembly in Pulmonary Arterial Hypertension.

The role of stress granules (SGs) in pulmonary arterial hypertension (PAH) is unknown. We hypothesized that SG formation contributes to abnormal vascular phenotypes, and cardiac and skeletal muscle dysfunction in PAH. Using the rat Sugen/hypoxia (SU/Hx) model of PAH, we demonstrate the formation of SG puncta and increased expression of SG proteins compared to control animals in lungs, right ventricles, and soleus muscles. Acetazolamide (ACTZ) treatment ameliorated the disease and reduced SG formation in all of these tissues. Primary pulmonary artery smooth muscle cells (PASMCs) from diseased animals had increased SG protein expression and SG number after acute oxidative stress and this was ameliorated by ACTZ. Pharmacologic inhibition of SG formation or genetic ablation of the SG assembly protein (G3BP1) altered the SU/Hx-PASMC phenotype by decreasing proliferation, increasing apoptosis and modulating synthetic and contractile marker expression. In human PAH lungs, we found increased SG puncta in pulmonary arteries compared to control lungs and in human PAH-PASMCs we found increased SGs after acute oxidative stress compared to healthy PASMCs. Genetic ablation of G3BP1 in human PAH-PASMCs resulted in a phenotypic switch to a less synthetic and more contractile phenotype. We conclude that increased SG formation in PASMCs and other tissues may contribute to PAH pathogenesis.

Activation of the mitochondrial unfolded protein response regulates the dynamic formation of stress granules.

To rapidly adapt to harmful changes to their environment, cells activate the integrated stress response (ISR). This results in an adaptive transcriptional and translational rewiring, and the formation of biomolecular condensates named stress granules (SGs), to resolve stress. In addition to this first line of defence, the mitochondrial unfolded protein response (UPRmt) activates a specific transcriptional programme to maintain mitochondrial homeostasis. We present evidence that SGs and UPRmt pathways are intertwined and communicate. UPRmt induction results in eIF2a phosphorylation and the initial and transient formation of SGs, which subsequently disassemble. The induction of GADD34 during late UPRmt protects cells from prolonged stress by impairing further assembly of SGs. Furthermore, mitochondrial functions and cellular survival are enhanced during UPRmt activation when SGs are absent, suggesting that UPRmt-induced SGs have an adverse effect on mitochondrial homeostasis. These findings point to a novel crosstalk between SGs and the UPRmt that may contribute to restoring mitochondrial functions under stressful conditions.

Stress granules formation in HEI-OC1 auditory cells and in H4 human neuroglioma cells secondary to cisplatin exposure

Abstract Stress granules (SGs) are highly dynamic micromolecular membraneless condensates that generate in cells subjected to stress. Formed from pools of untranslating messenger ribonucleoproteins (RNP), SGs dynamics constitute vital processes essential for cell survival. Here, we investigate whether established cytotoxic agents, such as the platinum-based chemotherapeutic agent cisplatin and the aminoglycoside antibiotic gentamicin, elicit SG formation in the House Ear Institute-Organ of Corti-1 (HEI-OC1) auditory cell line, H4 human neuroglioma cells and HEK-293T human embryonic kidney cells. Cells were treated with cisplatin or gentamicin for specific durations at designated concentrations. SG formation was assessed using immunocytochemistry and live cell imaging. Levels of essential proteins involved in SG assembly were evaluated using immunoblotting. We observed cisplatin-associated SG assembly in HEI-OC1 and H4 cells via confocal microscopy through antibody colabeling of G3BP1 with PABP or Caprin1. While maintaining an unchanged pattern of expression of main constituent SG proteins, cisplatin-related SGs in H4 cells persisted for at least 12 h after drug removal. Cells subjected to gentamicin exposure did not exhibit SGs. Our findings offer insights into subcellular mechanisms related to cisplatin-associated cytotoxicity, highlighting the need for future studies to further investigate this stress-response mechanism.

Myosin-5a facilitates stress granule formation by interacting with G3BP1

Stress granules (SGs) are non-membranous organelles composed of mRNA and proteins that assemble in the cytosol when the cell is under stress. Although the composition of mammalian SGs is both cell-type and stress-dependent, they consistently contain core components, such as Ras GTPase activating protein SH3 domain binding protein 1 (G3BP1). Upon stress, living cells rapidly assemble micrometric SGs, sometimes within a few minutes, suggesting that SG components may be actively transported by the microtubule and/or actin cytoskeleton. Indeed, SG assembly has been shown to depend on the microtubule cytoskeleton and the associated motor proteins. However, the role of the actin cytoskeleton and associated myosin motor proteins remains controversial. Here, we identified G3BP1 as a novel binding protein of unconventional myosin-5a (Myo5a). G3BP1 uses its C-terminal RNA-binding domain to interact with the middle portion of Myo5a tail domain (Myo5a-MTD). Suppressing Myo5a function in mammalian cells, either by overexpressing Myo5a-MTD, eliminating Myo5a gene expression, or treatment with myosin-5 inhibitor, inhibits the arsenite-induced formation of both small and large SGs. This is different from the effect of microtubule disruption, which abolishes the formation of large SGs but enhances the formation of small SGs under stress conditions. We therefore propose that, under stress conditions, Myo5a facilitates the formation of SGs at an earlier stage than the microtubule-dependent process.

Atomistic Insights into Sequence-Mediated Spontaneous Association of Short RNA Chains.

RNA-RNA association and phase separation appear to be essential for the assembly of stress granules and underlie RNA foci formation in repeat expansion disorders. RNA molecules are found to play a significant role in gene-regulatory functions via condensate formation among themselves or with RNA-binding proteins. The interplay between driven versus spontaneous processes is likely to be an important factor for controlling the formation of RNA-mediated biomolecular condensate. However, the sequence-specific interactions and molecular mechanisms that drive the spontaneous RNA-RNA association and help to form RNA-mediated phase-separated condensate remain unclear. With microseconds-long atomistic molecular simulations here, we report how essential aspects of RNA chains, namely, base composition, metal ion binding, and hydration properties, contribute to the association of the series of simplest biologically relevant homopolymeric and heteropolymeric short RNA chains. We show that spontaneous processes make the key contributions governed by the sequence-intrinsic properties of RNA chains, where the definite roles of base-specific hydrogen bonding and stacking interactions are prominent in the association of the RNA chains. Purine versus pyrimidine contents of RNA chains can directly influence the association properties of RNA chains by modulating hydrogen bonding and base stacking interactions. This study determines the impact of ionic environment in sequence-specific spontaneous association of short RNA chains, hydration features, and base-specific interactions of Na+, K+, and Mg2+ ions with RNA chains.

G3BP isoforms differentially affect stress granule assembly and gene expression during cellular stress.

Stress granules (SGs) are macromolecular assemblies that form under cellular stress. Formation of these membrane-less organelles is driven by the condensation of RNA and RNA-binding proteins such as G3BPs. G3BPs form SGs following stress-induced translational arrest. Three G3BP paralogs (G3BP1, G3BP2A, and G3BP2B) have been identified in vertebrates. However, the contribution of different G3BP paralogs to stress granule formation and gene expression changes is incompletely understood. Here, we probed the functions of G3BPs by identifying important residues for stress granule assembly at their N-terminal domain such as V11. This conserved amino acid is required for formation of the G3BP-Caprin-1 complex, hence promoting SG assembly. Total RNA sequencing and ribosome profiling revealed that a G3BPV11A mutant leads to changes in mRNA levels and ribosome engagement during the integrated stress response (ISR). Moreover, we found that G3BP2B preferentially forms stress granules and promotes changes in mRNA expression under endoplasmic reticulum (ER) stress. Furthermore, our work is a resource for researchers to study gene expression changes under cellular stress. Together, this work suggests that perturbing protein-protein interactions mediated by G3BPs affect stress granule assembly and gene expression during the ISR, and such functions are differentially regulated by G3BP paralogs under ER stress.

CoCl2-mimicked hypoxia induces the assembly of stress granules in trophoblast cells via eIF2α phosphorylation-dependent and -independent pathways.

Hypoxia has been implicated in preeclampsia (PE) pathophysiology. Stress granules (SGs) are present in the placenta of patients with PE. However, the pathways that contribute to SG aggregation in PE remain poorly understood. The objective of the current study is to investigate this issue. We first established an in vitro hypoxia model using human trophoblast cell line HTR-8/SVneo treated with cobalt chloride (CoCl2). CCK8 assay and wound healing assay were conducted to assess the viability and migration of HTR-8/SVneo cells after exposure to CoCl2-mimicked hypoxia. SG component expression in HTR-8/SVneo cells treated with CoCl2 alone, or in combination with indicated siRNAs was evaluated by reverse transcription quantitative PCR (RT-qPCR), western blot and immunofluorescence staining. Our results found CoCl2-mimicked hypoxia inhibits the proliferation and migration of HTR-8/SVneo cells. The treatment of CoCl2 can induce SG assembly in HTR-8/Svneo cells. Mechanistically, both heme-regulated inhibitors (HRI) mediated eukaryotic translation initiation factor (eIF)2α phosphorylation pathway and 4E binding protein 1 (4EBP1) pathway are involved in SG formation under the stress of CoCl2-mimicked hypoxia. Hypoxia-induced SGs in trophoblast cells might contribute to the etiology of PE.

Enhancement of Stress Granule Formation by a Chiral Compound Targeting G3BP1 via eIF2α Phosphorylation.

The chirality of a chemical differentiates it from its mirror-image counterpart. This unique property has significant implications in chemistry, biology, and drug discovery, where chiral chemicals display high selectivity and activity in achieving target specificity and reducing attrition rates in drug development. Stress granules (SGs) are dynamic assemblies of proteins and RNA that form in the cytoplasm of cells under stress conditions. Modulating their formation or disassembly could offer a novel approach to treating a wide range of diseases. This has led to significant interest in SGs as potential therapeutic targets. This study examined the NTF2-like domain of G3BP1 as a possible target for SG modulation. Molecular docking was used to simulate the interactions of compounds with the domain, and a potential candidate with a chiral structure was identified. The experiments showed that the compound induced the formation of SG-like granules. Importantly, the ability of this compound to modulate SG offers valuable insights into a new mechanism underlying the dynamics and promoting the assembly of SGs, and this new mechanism, in turn, holds potential for the development of drugs with diverse mechanisms of action and potentially synergistic effects.

A PARP14/TARG1-Regulated RACK1 MARylation Cycle Drives Stress Granule Dynamics in Ovarian Cancer Cells.

Mono(ADP-ribosyl)ation (MARylation) is emerging as a critical regulator of ribosome function and translation. Herein, we demonstrate that RACK1, an integral component of the ribosome, is MARylated on three acidic residues by the mono(ADP-ribosyl) transferase (MART) PARP14 in ovarian cancer cells. MARylation of RACK1 is required for stress granule formation and promotes the colocalization of RACK1 in stress granules with G3BP1, eIF3η, and 40S ribosomal proteins. In parallel, we observed reduced translation of a subset of mRNAs, including those encoding key cancer regulators (e.g., AKT). Treatment with a PARP14 inhibitor or mutation of the sites of MARylation on RACK1 blocks these outcomes, as well as the growth of ovarian cancer cells in culture and in vivo. To re-set the system after prolonged stress and recovery, the ADP-ribosyl hydrolase TARG1 deMARylates RACK1, leading to the dissociation of the stress granules and the restoration of translation. Collectively, our results demonstrate a therapeutically targetable pathway that controls stress granule assembly and disassembly in ovarian cancer cells.

Metal-polyphenol-network coated R612F nanoparticles reduce drug resistance in hepatocellular carcinoma by inhibiting stress granules

Stress granules (SGs) are considered to be the nonmembrane discrete assemblies present in the cytoplasm to cope with various environmental stress. SGs can promote the progression and drug resistance of hepatocellular carcinoma (HCC). Therefore, it is important to explore the mechanism of SG formation to reduce drug resistance in HCC. In this study, we demonstrate that p110α is required for SGs assembly. Mechanistically, the Arg-Gly (RG) motif of p110α is required for SG competence and regulates the recruitment of SG components. The methylation of p110α mediated by protein arginine methyltransferase 1 (PRMT1) interferes with the recruitment of p110α to SG components, thereby inhibiting the promotion of p110α to SGs. On this basis, we generated metal-polyphenol-network-coated R612F nanoparticles (MPN-R612F), which can efficiently enter HCC cells and maintain the hypermethylation state of p110α, thereby inhibiting the assembly of SGs and ultimately reducing the resistance of HCC cells to sorafenib. The combination of MPN-R612F nanoparticles and sorafenib can kill HCC cells more effectively and play a stronger anti-tumor effect. This study provides a new perspective for targeting SGs in the treatment of HCC.

The network interactions between the porcine deltacoronavirus nucleocapsid protein and host cellular proteins

Porcine deltacoronavirus (PDCoV) is an emerging swine coronavirus that can cause diarrhea in pigs of all ages with varying severity. Host–virus protein interactions are critical for intracellular viral replication. Elucidating the interactions between cellular and viral proteins can help us to design antiviral strategies. PDCoV N protein is the most abundant and vital regulator in virus replication. In this study, 604 host proteins were identified to interact with PDCoV N protein by Co-IP combined with LC-MS, of which 243 proteins were specifically bound to N protein. PPI analysis revealed that the N-interacting host proteins are categorized into three groups: ribonucleoprotein complex biogenesis modulation, cellular nitrogen compound metabolism, and nucleic acid binding. GO and KEGG analyses showed that the host proteins are primarily involved in mRNA splicing, stress granule assembly, spliceosomal snRNP assembly. Additionally, four host proteins-TRIM25, HNRNPUL1, RPS27A, and SLC3A2-were selected to validate the interactome data through Co-IP and Confocal assays. This study can help in designing anti-PDCoV strategies and understanding the replication mechanism of PDCoV.

PRMT1 and TDRD3 promote stress granule assembly by rebuilding the protein-RNA interaction network

Stress granules (SGs) are membrane-less organelles (MLOs) or cytosolic compartments formed upon exposure to environmental cell stress-inducing stimuli. SGs are based on ribonucleoprotein complexes from a set of cytoplasmic proteins and mRNAs, blocked in translation due to stress cell-induced polysome disassembly. Post-translational modifications (PTMs) such as methylation, are involved in SG assembly, with the methylation writer PRMT1 and its reader TDRD3 colocalizing to SGs. However, the role of this writer-reader system in SG assembly remains unclear. Here, we found that PRMT1 methylates SG constituent RNA-binding proteins (RBPs) on their RGG motifs. Besides, we report that TDRD3, as a reader of asymmetric dimethylarginines, enhances RNA binding to recruit additional RNAs and RBPs, lowering the percolation threshold and promoting SG assembly. Our study enriches our understanding of the molecular mechanism of SG formation by elucidating the functions of PRMT1 and TDRD3. We anticipate that our study will provide a new perspective for comprehensively understanding the functions of PTMs in liquid-liquid phase separation driven condensate assembly.

Myeloid PTEN loss affects the therapeutic response by promoting stress granule assembly and impairing phagocytosis by macrophages in breast cancer

Breast cancer (BRCA) has become the most common type of cancer in women. Improving the therapeutic response remains a challenge. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a classic tumour suppressor with emerging new functions discovered in recent years, and myeloid PTEN loss has been reported to impair antitumour immunity. In this study, we revealed a novel mechanism by which myeloid PTEN potentially affects antitumour immunity in BRCA. We detected accelerated stress granule (SG) assembly under oxidative stress in PTEN-deficient bone marrow-derived macrophages (BMDMs) through the EGR1-promoted upregulation of TIAL1 transcription. PI3K/AKT/mTOR (PAM) pathway activation also promoted SG formation. ATP consumption during SG assembly in BMDMs impaired the phagocytic ability of 4T1 cells, potentially contributing to the disruption of antitumour immunity. In a BRCA neoadjuvant cohort, we observed a poorer response in myeloid PTENlow patients with G3BP1 aggregating as SGs in CD68+ cells, a finding that was consistent with the observation in our study that PTEN-deficient macrophages tended to more readily assemble SGs with impaired phagocytosis. Our results revealed the unconventional impact of SGs on BMDMs and might provide new perspectives on drug resistance and therapeutic strategies for the treatment of BRCA patients.

Caprin1 Bridges PRMT1 to G3BP1 and Spaces Them to Ensure Proper Stress Granule Formation

Stress granules (SGs) are dynamic biomolecular condensates that form in the cytoplasm in response to cellular stress, encapsulating proteins and RNAs. Methylation is a key factor in the assembly of SGs, with PRMT1, which acts as an arginine methyltransferase, localizing to SGs. However, the precise mechanism of PRMT1 localization within SGs remains unknown. In this study, we identified that Caprin1 plays a primary role in the recruitment of PRMT1 to SGs, particularly through its C-terminal domain. Our findings demonstrate that Caprin1 serves a dual function as both a linker, facilitating the formation of a PRMT1-G3BP1 complex, and as a spacer, preventing the aberrant formation of SGs under non-stress conditions. This study sheds new lights on the regulatory mechanisms governing SG formation and suggests that Caprin1 plays a critical role in cellular responses to stress.

Replication properties of a contemporary Zika virus from West Africa.

Zika virus (ZIKV) has become a global health problem over the past decade due to the extension of the geographic distribution of the Asian/American genotype. Recent epidemics of Asian/American ZIKV have been associated with developmental disorders in humans. There is mounting evidence that African ZIKV may be associated with increased fetal pathogenicity necessitating to pay a greater attention towards currently circulating viral strains in sub-Saharan Africa. Here, we generated an infectious molecular clone GUINEA-18 of a recently transmitted human ZIKV isolate from West Africa, ZIKV-15555. The available infectious molecular clone MR766MC of historical African ZIKV strain MR766-NIID was used for a molecular clone-based comparative study. Viral clones GUINEA-18 and MR766MC were compared for their ability to replicate in VeroE6, A549 and HCM3 cell lines. There was a lower replication rate for GUINEA-18 associated with weaker cytotoxicity and reduced innate immune system activation compared with MR766MC. Analysis of chimeric viruses between viral clones stressed the importance of NS1 to NS4B proteins, with a particular focus of NS4B on GUINEA-18 replicative properties. ZIKV has developed strategies to prevent cytoplasmic stress granule formation which occurs in response to virus infection. GUINEA-18 was greatly efficient in inhibiting stress granule assembly in A549 cells subjected to a physiological stressor, with NS1 to NS4B proteins also being critical in this process. The impact of these GUINEA-18 proteins on viral replicative abilities and host-cell responses to viral infection raises the question of the role of nonstructural proteins in the pathogenicity of currently circulating ZIKV in sub-Saharan Africa.

USP21-mediated G3BP1 stabilization accelerates proliferation and metastasis of esophageal squamous cell carcinoma via activating Wnt/β-Catenin signaling

Lacking effective therapeutic targets heavily restricts the improvement of clinical prognosis for patients diagnosed with esophageal squamous cell carcinoma (ESCC). Ubiquitin Specific Peptidase 21 (USP21) is dysregulated in plenty of human cancers, however, its potential function and relevant molecular mechanisms in ESCC malignant progression as well as its value in clinical translation remain largely unknown. Here, in vitro and in vivo experiments revealed that aberrant upregulation of USP21 accelerated the proliferation and metastasis of ESCC in a deubiquitinase-dependent manner. Mechanistically, we found that USP21 binds to, deubiquitinates, and stabilizes the G3BP Stress Granule Assembly Factor 1 (G3BP1) protein, which is required for USP21-mediated ESCC progression. Further molecular studies demonstrated that the USP21/G3BP1 axis played a tumor-promoting role in ESCC progression by activating the Wnt/β-Catenin signaling pathway. Additionally, disulfiram (DSF), an inhibitor against USP21 deubiquitylation activity, markedly abolished the USP21-mediated stability of G3BP1 protein and significantly displayed an anti-tumor effect on USP21-driving ESCC progression. Finally, the regulatory axis of USP21/G3BP1 was demonstrated to be aberrantly activated in ESCC tumor tissues and closely associated with advanced clinical stages and unfavorable prognoses, which provides a promising therapeutic strategy targeting USP21/G3BP1 axis for ESCC patients.

ZO-1 interacts with YB-1 in endothelial cells to regulate stress granule formation during angiogenesis

Zonula occludens-1 (ZO-1) is involved in the regulation of cell-cell junctions between endothelial cells (ECs). Here we identify the ZO-1 protein interactome and uncover ZO-1 interactions with RNA-binding proteins that are part of stress granules (SGs). Downregulation of ZO-1 increased SG formation in response to stress and protected ECs from cellular insults. The ZO-1 interactome uncovered an association between ZO-1 and Y-box binding protein 1 (YB-1), a constituent of SGs. Arsenite treatment of ECs decreased the interaction between ZO-1 and YB-1, and drove SG assembly. YB-1 expression is essential for SG formation and for the cytoprotective effects induced by ZO-1 downregulation. In the developing retinal vascular plexus of newborn mice, ECs at the front of growing vessels express less ZO-1 but display more YB-1-positive granules than ECs located in the vascular plexus. Endothelial-specific deletion of ZO-1 in mice at post-natal day 7 markedly increased the presence of YB-1-positive granules in ECs of retinal blood vessels, altered tip EC morphology and vascular patterning, resulting in aberrant endothelial proliferation, and arrest in the expansion of the retinal vasculature. Our findings suggest that, through its interaction with YB-1, ZO-1 controls SG formation and the response of ECs to stress during angiogenesis.

NtG3BPL1 confers resistance to chilli veinal mottle virus through promoting the degradation of 6K2 in tobacco.

The RNA regulatory network is a complex and dynamic regulation in plant cells involved in mRNA modification, translation, and degradation. Ras-GAP SH3 domain-binding protein (G3BP) is a scaffold protein for the assembly of stress granules (SGs) and is considered an antiviral component in mammals. However, the function of G3BP during virus infection in plants is still largely unknown. In this study, four members of the G3BP-like proteins (NtG3BPLs) were identified in Nicotiana tabacum and the expression levels of NtG3BPL1 were upregulated during chilli veinal mottle virus (ChiVMV) infection. NtG3BPL1 was localized in the nucleus and cytoplasm, forming cytoplasmic granules under transient high-temperature treatment, whereas the abundance of cytoplasmic granules was decreased under ChiVMV infection. Overexpression of NtG3BPL1 inhibited ChiVMV infection and delayed the onset of symptoms, whereas knockout of NtG3BPL1 promoted ChiVMV infection. In addition, NtG3BPL1 directly interacted with ChiVMV 6K2 protein, whereas 6K2 protein had no effect on NtG3BPL1-derived cytoplasmic granules. Further studies revealed that the expression of NtG3BPL1 reduced the chloroplast localization of 6K2-GFP and the NtG3BPL1-6K2 interaction complex was localized in the cytoplasm. Furthermore, NtG3BPL1 promoted the degradation of 6K2 through autophagy pathway, and the accumulation of 6K2 and ChiVMV was affected by autophagy activation or inhibition in plants. Taken together, our results demonstrate that NtG3BPL1 plays a positive role in tobacco resistance against ChiVMV infection, revealing a novel mechanism of plant G3BP in antiviral strategy.

Mammalian IRE1α dynamically and functionally coalesces with stress granules.

Upon endoplasmic reticulum (ER) stress, activation of the ER-resident transmembrane protein kinase/endoribonuclease inositol-requiring enzyme 1 (IRE1) initiates a key branch of the unfolded protein response (UPR) through unconventional splicing generation of the transcription factor X-box-binding protein 1 (XBP1s). Activated IRE1 can form large clusters/foci, whose exact dynamic architectures and functional properties remain largely elusive. Here we report that, in mammalian cells, formation of IRE1α clusters is an ER membrane-bound phase separation event that is coupled to the assembly of stress granules (SGs). In response to different stressors, IRE1α clusters are dynamically tethered to SGs at the ER. The cytosolic linker portion of IRE1α possesses intrinsically disordered regions and is essential for its condensation with SGs. Furthermore, disruption of SG assembly abolishes IRE1α clustering and compromises XBP1 mRNA splicing, and such IRE1α-SG coalescence engenders enrichment of the biochemical components of the pro-survival IRE1α-XBP1 pathway during ER stress. Our findings unravel a phase transition mechanism for the spatiotemporal assembly of IRE1α-SG condensates to establish a more efficient IRE1α machinery, thus enabling higher stress-handling capacity.