Component Of Stress Granules Research Articles

Dynamic composition of stress granules in Trypanosoma brucei.

Stress granules (SGs) are stress-induced RNA condensates consisting of stalled initiation complexes resulting from translational inhibition. The biochemical composition and function of SGs are highly diverse, and this diversity has been attributed to different stress conditions, signalling pathways involved and specific cell types. Interestingly, mRNA decay components, which are found in ubiquitous cytoplasmic foci known as processing bodies (PB), have also been identified in SG proteomes. A major challenge in current SG studies is to understand the cause of SG diversity, as well as the function of SG under different stress conditions. Trypanosoma brucei is a single-cellular parasite that causes Human African Trypanosomiasis (sleping sickness). In this study, we showed that by varying the supply of extracellular carbon sources during starvation, cellular ATP levels changed rapidly, resulting in SGs of different compositions and dynamics. We identified a subset of SG components, which dissociated from the SGs in response to cellular ATP depletion. Using expansion microscopy, we observed sub-granular compartmentalization of PB- and SG-components within the stress granules. Our results highlight the importance of cellular ATP in SG composition and dynamics, providing functional insight to SGs formed under different stress conditions.

Endogenous retrovirus-like proteins recruit UBQLN2 to stress granules and alter their functional properties.

The human genome is replete with sequences derived from foreign elements including endogenous retrovirus-like proteins of unknown function. Here we show that UBQLN2, a ubiquitin-proteasome shuttle factor implicated in neurodegenerative diseases, is regulated by the linked actions of two retrovirus-like proteins, RTL8 and PEG10. RTL8 confers on UBQLN2 the ability to complex with and regulate PEG10. PEG10, a core component of stress granules, drives the recruitment of UBQLN2 to stress granules under various stress conditions, but can only do so when RTL8 is present. Changes in PEG10 levels further remodel the kinetics of stress granule disassembly and overall composition by incorporating select extracellular vesicle proteins. Within stress granules, PEG10 forms virus-like particles, underscoring the structural heterogeneity of this class of biomolecular condensates. Together, these results reveal an unexpected link between pathways of cellular proteostasis and endogenous retrovirus-like proteins.

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.

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.

Unravelling stress granules in the deep cold: Characterisation of TIA-1 gene sequence in Antarctic fish species

Stress granules (SGs) are cytoplasmic foci lacking membranes, comprising non-translating messenger ribonucleoproteins, translational initiation factors, and additional proteins. Their formation is crucial for rapidly modulating gene expression in response to adverse environmental conditions, such as pollution and infections. Limited research has focused on investigating the molecular components of SGs in fish, with minimal exploration in Antarctic fish. This study characterises for the first time the transcript sequences of one key protein component of SGs, TIA-1 (T-cell intracellular antigen 1), in two Antarctic endemic fish species, i.e. Trematomus bernacchii and Chionodraco hamatus. The mRNA-binding protein TIA-1 acts as a post-transcriptional regulator of gene expression and its aggregation leads to the formation of SGs in response to cellular damage. The in vitro and bioinformatic analyses of the TIA-1 gene sequences of these two species highlighted interesting peculiarities, which include the transcription of alternatively spliced isoforms unique to the notothenioid lineage, potentially unlocking further insights into their unique adaptations to extreme environmental conditions. This is the first study to analyze tia-1 expression levels in different tissues of Antarctic fish species. Our key findings indicate that the TIA-1 gene is expressed at particularly high levels in the liver and spleen of C. hamatus, as well as in the heart and skeletal muscle of T. bernacchii. This suggests that those tissues play a significant role in the stress response mechanisms of the studied species. This study provides novel insights into the molecular adaptations of Antarctic fish, highlighting the potential importance of TIA-1 in their response to environmental stressors. The unique features of TIA-1 identified in these species may offer broader implications for understanding how Antarctic fish regulate gene transcriptions in their extreme environments.

G3BP2 promotes tumor progression and gemcitabine resistance in PDAC via regulating PDIA3-DKC1-hENT in a stress granules-dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) is distinguished by its aggressive malignancy, limited treatment avenues and a tendency towards chemotherapy resistance, underscoring the critical need for advanced research to uncover new therapeutic approaches. Stress granules (SGs) that is implicated in cellular self-protection mechanism, along with its associated family molecules have shown pro-cancer effects and are closely related to tumor chemotherapy resistance. In this study we investigated the relationship between Ras GTPase-activating protein-binding proteins 2 (G3BP2), a core component of SGs, and the malignancy of PDAC as well as its resistance to the chemotherapy drug gemcitabine. Analyzing TCGA dataset revealed that the expression of G3BP1 and G3BP2 was significantly upregulated in PDAC compared with adjacent normal pancreatic tissues, and the high expression of G3BP2 rather than G3BP1 was significantly associated with poorer overall survival (OS) in PDAC patients. We demonstrated that knockdown of G3BP2 inhibited the proliferation and invasion of PANC-1 and CFPAC-1 cells in vitro and in vivo. By analyzing the differentially expressed genes in G3BP2 knockdown and overexpressed PANC-1 cells, we identified DKC1 that was associated with RNA stability and regulation as the target of G3BP2. We demonstrated that G3BP2 bound to PDIA3 mRNA and recruited them into SGs, increasing the stability of PDIA3 mRNA and attenuating its translation efficiency, thereby promoting DKC1 expression. Furthermore, DKC1 could bind to hENT mRNA and inhibited its expression, which enhanced gemcitabine resistance of PDAC. Therefore, we propose a novel mechanism wherein G3BP2 facilitates PDAC's resistance to chemotherapy by modulating PDIA3-DKC1-hENT in a SGs-dependent way, suggesting G3BP2 SGs a protentional therapeutic target for the treatment in PDAC.

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.

Atomic resolution map of the solvent interactions driving SOD1 unfolding in CAPRIN1 condensates

Biomolecules can be sequestered into membrane-less compartments, referred to as biomolecular condensates. Experimental and computational methods have helped define the physical-chemical properties of condensates. Less is known about how the high macromolecule concentrations in condensed phases contribute "solvent" interactions that can remodel the free-energy landscape of other condensate-resident proteins, altering thermally accessible conformations and, in turn, modulating function. Here, we use solution NMR spectroscopy to obtain atomic resolution insights into the interactions between the immature form of superoxide dismutase 1 (SOD1), which can mislocalize and aggregate in stress granules, and the RNA-binding protein CAPRIN1, a component of stress granules. NMR studies of CAPRIN1:SOD1 interactions, focused on both unfolded and folded SOD1 states in mixed phase and demixed CAPRIN1-based condensates, establish that CAPRIN1 shifts the SOD1 folding equilibrium toward the unfolded state through preferential interactions with the unfolded ensemble, with little change to the structure of the folded conformation. Key contacts between CAPRIN1 and the H80-H120 region of unfolded SOD1 are identified, as well as SOD1 interaction sites near both the arginine-rich and aromatic-rich regions of CAPRIN1. Unfolding of immature SOD1 in the CAPRIN1 condensed phase is shown to be coupled to aggregation, while a more stable zinc-bound, dimeric form of SOD1 is less susceptible to unfolding when solvated by CAPRIN1. Our work underscores the impact of the condensate solvent environment on the conformational states of resident proteins and supports the hypothesis that ALS mutations that decrease metal binding or dimerization function as drivers of aggregation in condensates.

Proteasome resides in and dismantles plant heat stress granules constitutively

Stress granules (SGs) are conserved reversible cytoplasmic condensates enriched with aggregation-prone proteins assembled in response to various stresses. How plants regulate SG dynamics is unclear. Here, we show that 26S proteasome is a stable component of SGs, promoting the overall clearance of SGs without affecting the molecular mobility of SG components. Increase in either temperature or duration of heat stress reduces the molecular mobility of SG marker proteins and suppresses SG clearance. Heat stress induces dramatic ubiquitylation of SG components and enhances the activities of SG-resident proteasomes, allowing the degradation of SG components even during the assembly phase. Their proteolytic activities enable the timely disassembly of SGs and secure the survival of plant cells during the recovery from heat stress. Therefore, our findings identify the cellular process that de-couples macroscopic dynamics of SGs from the molecular dynamics of its constituents and highlights the significance of the proteasomes in SG disassembly.

Nanoscale dynamics and localization of single endogenous mRNAs in stress granules.

Stress granules (SGs) are cytoplasmic messenger ribonucleoprotein granules transiently formed in stressed mammalian cells. Although SG components have been well characterized, detailed insights into the molecular behavior inside SGs remain unresolved. We investigated nanoscale dynamics and localization of endogenous mRNAs in SGs combining single mRNA tracking and super-resolution localization microscopy. First, we developed a methodology for tracking single mRNAs within SGs, revealing that although mRNAs in SGs are mainly stationary (∼40%), they also move in a confined (∼25%) or freely diffusing (∼35%) manner. Second, the super-resolution localization microscopy showed that the mRNAs in SGs are heterogeneously distributed and partially form high-density clusters. Third, we simultaneously performed single mRNA tracking and super-resolution microscopy in SGs, demonstrating that single mRNA trajectories are mainly found around high-density clusters. Finally, a quantitative analysis of mRNA localization and dynamics during stress removal was conducted using live super-resolution imaging and single-molecule tracking. These results suggest that SGs have a highly organized structure that enables dynamic regulation of the mRNAs at the nanoscale, which is responsible for the ordered formation and the wide variety of functions of SGs.

UBAP2L contributes to formation of P-bodies and modulates their association with stress granules.

Stress triggers the formation of two distinct cytoplasmic biomolecular condensates: stress granules (SGs) and processing bodies (PBs), both of which may contribute to stress-responsive translation regulation. Though PBs can be present constitutively, stress can increase their number and size and lead to their interaction with stress-induced SGs. The mechanism of such interaction, however, is largely unknown. Formation of canonical SGs requires the RNA binding protein Ubiquitin-Associated Protein 2-Like (UBAP2L), which is a central SG node protein in the RNA-protein interaction network of SGs and PBs. UBAP2L binds to the essential SG and PB proteins G3BP and DDX6, respectively. Research on UBAP2L has mostly focused on its role in SGs, but not its connection to PBs. We find that UBAP2L is not solely an SG protein but also localizes to PBs in certain conditions, contributes to PB biogenesis and SG-PB interactions, and can nucleate hybrid granules containing SG and PB components in cells. These findings inform a new model for SG and PB formation in the context of UBAP2L's role.

Concerted action of ataxin-2 and PABPC1-bound mRNA poly(A) tail in the formation of stress granules.

Stress induces global stabilization of the mRNA poly(A) tail (PAT) and the assembly of untranslated poly(A)-tailed mRNA into mRNPs that accumulate in stress granules (SGs). While the mechanism behind stress-induced global PAT stabilization has recently emerged, the biological significance of PAT stabilization under stress remains elusive. Here, we demonstrate that stress-induced PAT stabilization is a prerequisite for SG formation. Perturbations in PAT length impact SG formation; PAT shortening, achieved by overexpressing mRNA deadenylases, inhibits SG formation, whereas PAT lengthening, achieved by overexpressing their dominant negative mutants or downregulating deadenylases, promotes it. PABPC1, which specifically binds to the PAT, is crucial for SG formation. Complementation analyses reveal that the PABC/MLLE domain of PABPC1, responsible for binding PAM2 motif-containing proteins, plays a key role. Among them, ataxin-2 is a known SG component. A dominant-negative approach reveals that the PAM2 motif of ataxin-2 is essential for SG formation. Notably, ataxin-2 increases stress sensitivity, lowering the threshold for SG formation, probably by promoting the aggregation of PABPC1-bound mRNA. The C-terminal region is responsible for the self-aggregation of ataxin-2. These findings underscore the critical roles of mRNA PAT, PABPC1 and ataxin-2 in SG formation and provide mechanistic insights into this process.

Heart-specific NFAT5 knockout suppresses type I interferon signaling and aggravates coxsackievirus-induced myocarditis.

Nuclear factor of activated T cells 5 (NFAT5) is an osmosensitive transcription factor that is well-studied in renal but rarely explored in cardiac diseases. Although the association of Coxsackievirus B3 (CVB3) with viral myocarditis is well-established, the role of NFAT5 in this disease remains largely unexplored. Previous research has demonstrated that NFAT5 restricts CVB3 replication yet is susceptible to cleavage by CVB3 proteases. Using an inducible cardiac-specific Nfat5-knockout mouse model, we uncovered that NFAT5-deficiency exacerbates cardiac pathology, worsens cardiac function, elevates viral load, and reduces survival rates. RNA-seq analysis of CVB3-infected mouse hearts revealed the significant impact of NFAT5-deficiency on gene pathways associated with cytokine signaling and inflammation. Subsequent in vitro and in vivo investigation validated the disruption of the cytokine signaling pathway in response to CVB3 infection, evidenced by reduced expression of key cytokines such as interferon β1 (IFNβ1), C-X-C motif chemokine ligand 10 (CXCL10), interleukin 6 (IL6), among others. Furthermore, NFAT5-deficiency hindered the formation of stress granules, leading to a reduction of important stress granule components, including plakophilin-2, a pivotal protein within the intercalated disc, thereby impacting cardiomyocyte structure and function. These findings unveil a novel mechanism by which NFAT5 inhibits CVB3 replication and pathogenesis through the promotion of antiviral type I interferon signaling and the formation of cytoplasmic stress granules, collectively identifying NFAT5 as a new cardio protective protein.

TRIM25 predominately associates with anti-viral stress granules

Stress granules (SGs) are induced by various environmental stressors, resulting in their compositional and functional heterogeneity. SGs play a crucial role in the antiviral process, owing to their potent translational repressive effects and ability to trigger signal transduction; however, it is poorly understood how these antiviral SGs differ from SGs induced by other environmental stressors. Here we identify that TRIM25, a known driver of the ubiquitination-dependent antiviral innate immune response, is a potent and critical marker of the antiviral SGs. TRIM25 undergoes liquid-liquid phase separation (LLPS) and co-condenses with the SG core protein G3BP1 in a dsRNA-dependent manner. The co-condensation of TRIM25 and G3BP1 results in a significant enhancement of TRIM25’s ubiquitination activity towards multiple antiviral proteins, which are mainly located in SGs. This co-condensation is critical in activating the RIG-I signaling pathway, thus restraining RNA virus infection. Our studies provide a conceptual framework for better understanding the heterogeneity of stress granule components and their response to distinct environmental stressors.

The Role of Heat-Induced Stress Granules in the Blood-Testis Barrier of Mice.

Stress granules (SGs) are membraneless ribonucleoprotein (RNP)-based cellular foci formed in response to stress, facilitating cell survival by protecting against damage. Mammalian spermatogenesis should be maintained below body temperature for proper development, indicating its vulnerability to heat stress (HS). In this study, biotin tracer permeability assays showed that the inhibition of heat-induced SG assembly in the testis by 4-8 mg/kg cycloheximide significantly increased the percentage of seminiferous tubules with a damaged blood-testis barrier (BTB). Western blot results additionally revealed that the suppression of heat-induced SG assembly in Sertoli cell line, TM4 cells, by RNA inference of G3bp1/2 aggravated the decline in the BTB-related proteins ZO-1, β-Catenin and Claudin-11, indicating that SGs could protect the BTB against damage caused by HS. The protein components that associate with SGs in Sertoli cells were isolated by sequential centrifugation and immunoprecipitation, and were identified by liquid chromatography with tandem mass spectrometry. Gene Ontology and KEGG pathway enrichment analysis revealed that their corresponding genes were mainly involved in pathways related to proteasomes, nucleotide excision repair, mismatch repair, and DNA replication. Furthermore, a new SG component, the ubiquitin associated protein 2 (UBAP2), was found to translocate to SGs upon HS in TM4 cells by immunofluorescence. Moreover, SG assembly was significantly diminished after UBAP2 knockdown by RNA inference during HS, suggesting the important role of UBAP2 in SG assembly. In addition, UBAP2 knockdown reduced the expression of ZO-1, β-Catenin and Claudin-11, which implied its potential role in the function of the BTB. Overall, our study demonstrated the role of SGs in maintaining BTB functions during HS and identified a new component implicated in SG formation in Sertoli cells. These findings not only offer novel insights into the biological functions of SGs and the molecular mechanism of low fertility in males in summer, but also potentially provide an experimental basis for male fertility therapies.

Abstract P03: Splicing factor mutations induce stress granule upregulation in myeloid malignancies

Abstract Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are aggressive blood cancers with a 5-year relative survival rate of only 37% and 28%, respectively [Sekeres MA, Taylor J. JAMA. 2022; https://www.cancer.net/cancer-types/leukemia-acute-myeloid-aml/statistics]. This highlights the need to better characterize the crucial oncogenic mechanisms sustaining cancer stem cell fitness in order to develop more effective therapies. Somatic mutations affecting splicing factor genes represent a hallmark of myeloid blood cancers [Saez B, et al. Blood. 2017]. Splicing factor mutations alter the splicing process of thousands of RNA molecules, but how they provide clonal advantage with consequent ineffective hematopoiesis remains an unanswered question. We integrated fractionated enhanced UV-crosslinking and immunoprecipitation (freCLIP-seq), bulk RNA-seq and immunofluorescence imaging demonstrating that U2AF1 mutations in myeloid malignancies alter the biology of stress granules (SGs), whose upregulation has been linked to tumorigenesis [Redding A, Grabocka E. Trends Cancer. 2023]. U2AF1 mutations, through RNA binding and splicing alterations, increase the availability of SG components leading to enhanced stress adaptation. Crucially, U2AF1-mutant cells show improved cell fitness that can be reverted by a SG inhibitor [Biancon G, et al. Mol Cell. 2022]. These observations have been recently validated at single-cell level in MDS patients (n=10) without splicing factor mutations, with U2AF1 S34F mutation, or with SRSF2 P95H mutation that carries an equally poor prognosis as U2AF1-mutant disease. We isolated CD34+ blasts from bone marrow samples and performed 10x Genomics 5' scRNA-seq. Integrating cell type annotation (https://github.com/TebaldiLab/TheCellMarkerAccordion) with mutational analysis (VarTrix v1.1.19), we clearly distinguished an enrichment of U2AF1 mutant cells and of SRSF2 mutant cells in hematopoietic multipotent progenitor cells. The enrichment is observed also in monocytic cells that are also characterized by high SG score. Of note, it has been recently showed that leukemic stem cells with monocytic phenotype are associated to relapse in AML patients treated with BCL2-inh [Pei S, et al. Cancer Discov. 2023]. We then identified the top influential SG genes in each annotated mutant population and conducted functional enrichment analysis. In hematopoietic multipotent progenitor cells, the biological processes mainly affected by the SG upregulation are regulation of translation, of microtubule, of transcription and of cell growth. Changes in SGs may represent the collective result of splicing aberrancies that drive ineffective hematopoiesis in MDS and AML, or other potential tumorigenic phenotypes. Splicing factor mutations are in fact reported in several cancer types [Stanley RF, Abdel-Wahab O. Nat Cancer. 2022] and SGs are involved in several cancer hallmarks [Boija A, et al. Cancer Cell. 2021]. Alterations in SGs provide therefore novel therapeutic opportunities in blood cancers and potentially in other cancers. Citation Format: Giulia Biancon, Emma Busarello, Matthew Cheng, Domitilla Baccon, Jennifer VanOudenhove, Fabio Ciceri, Toma Tebaldi, Stephanie Halene. Splicing factor mutations induce stress granule upregulation in myeloid malignancies [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P03.

Heat stress promotes Arabidopsis AGO1 phase separation and association with stress granule components

In Arabidopsis thaliana, ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing. AGO1 associates to the rough endoplasmic reticulum to conduct miRNA-mediated translational repression, mRNA cleavage, and biogenesis of phased siRNAs. Here, we show that a 37°C heat stress (HS) promotes AGO1 protein accumulation in cytosolic condensates where it colocalizes with components of siRNA bodies and of stress granules. AGO1 contains a prion-like domain in its poorly characterized N-terminal Poly-Q domain, which is sufficient to undergo phase separation independently of the presence of SGS3. HS only moderately affects the small RNA repertoire, the loading of AGO1 by miRNAs, and the signatures of target cleavage, suggesting that its localization in condensates protects AGO1 rather than promoting or impairing its activity in reprogramming gene expression during stress. Collectively, our work sheds new light on the impact of high temperature on a main effector of RNA silencing in plants.

Formation of the NLRP3 inflammasome inhibits stress granule assembly by multiple mechanisms.

Proper regulation of cellular response to environmental stress is crucial for maintaining biological homeostasis and is achieved by the balance between cell death processes, such as the formation of the pyroptosis-inducing NLRP3 inflammasome, and pro-survival processes, such as stress granule (SG) assembly. However, the functional interplay between these two stress-responsive organelles remains elusive. Here, we identified DHX33, a viral RNA sensor for the NLRP3 inflammasome, as a SG component, and the SG-nucleating protein G3BP as an NLRP3 inflammasome component. We also found that a decrease in intracellular potassium (K+) concentration, a key common step in NLRP3 inflammasome activation, markedly inhibited SG assembly. Therefore, when macrophages are exposed to stress stimuli with the potential to induce both SGs and the NLRP3 inflammasome, such as cytoplasmic poly(I:C) stimulation, they preferentially form the NLRP3 inflammasome but avoid SG assembly by sequestering G3BP into the inflammasome and by inducing a reduction in intracellular K+ levels. Thus, under such conditions, DHX33 is primarily utilized as a viral RNA sensor for the inflammasome. Our data reveal the functional crosstalk between NLRP3 inflammasome-mediated pyroptosis and SG-mediated cell survival pathways, and delineate a molecular mechanism that regulates cell-fate decisions and anti-viral innate immunity under stress.

YBX1 Underwent Phase Separation into Stress Granules Stimulated by Ionizing Radiation.

Stress granules (SGs) are formed through liquid-liquid phase separation (LLPS), in response to external stimuli. YBX1, an integral component of SGs, plays a crucial role in tumor progression and cellular stress response. This study aims to elucidate the mechanisms and specific biological implications of YBX1 in SG formation, along with the identification of key regions and interacting proteins. Our observations indicate that YBX1 rapidly undergoes liquid-liquid phase separation, leading to SG formation in response to 8 Gy X-ray irradiation within 1 h, with SGs reverting to their original state after 5 h. There was a potential interaction between ATXN2L and YBX1, persisting YBX1 within the SGs. Our data suggested a potential interaction between ATXN2L and YBX1, and it remained associated with YBX1 within the SGs. Furthermore, our subsequent studies demonstrate that targeting ATXN2L can diminish the recruitment of YBX1 to stress granules (SGs), consequently enhancing the radiosensitivity of HeLa cells.

Stress granule-related genes during embryogenesis of an invertebrate chordate.

Controlling global protein synthesis through the assembly of stress granules represents a strategy adopted by eukaryotic cells to face various stress conditions. TIA 1-related nucleolysin (TIAR), tristetraprolin (TTP), and Ras-GTPase-activating protein SH3-domain-binding protein (G3BP) are key components of stress granules, allowing the regulation of mRNA stability, and thus controlling not only stress responses but also cell proliferation and differentiation. In this study, we aimed at investigating the roles of tiar, ttp, and g3bp during embryogenesis of the solitary ascidian Ciona robusta under both physiological and stress conditions. We carried out CRISPR/Cas9 to evaluate the effects of gene knockout on normal embryonic development, and gene reporter assay to study the time and tissue specificity of gene transcription, together with whole-mount in situ hybridization and quantitative real time PCR. To induce acute stress conditions, we used iron and cadmium as "essential" and "non-essential" metals, respectively. Our results highlight, for the first time, the importance of tiar, ttp, and g3bp in controlling the development of mesendodermal tissue derivatives during embryogenesis of an invertebrate chordate.