Stress granules are dispensable for mRNA stabilization during cellular stress

During cellular stress, protein synthesis is severely reduced and bulk mRNA is recruited to stress granules (SGs). Previously, we showed that the SG-recruited IGF2 mRNA-binding protein 1 (IGF2BP1) interferes with target mRNA degradation during cellular stress. Whether this requires the formation of SGs remained elusive. Here, we demonstrate that the sustained inhibition of visible SGs requires the concomitant knockdown of TIA1, TIAR and G3BP1. FRAP and photo-conversion studies, however, indicate that these proteins only transiently associate with SGs. This suggests that instead of forming a rigid scaffold for mRNP recruitment, TIA proteins and G3BP1 promote SG-formation by constantly replenishing mRNPs. In contrast, RNA-binding proteins like IGF2BP1 or HUR, which are dispensable for SG-assembly, are stably associated with SGs and the IGF2BP1/HUR-G3BP1 association is increased during stress. The depletion of IGF2BP1 enhances the degradation of target mRNAs irrespective of inhibiting SG-formation, whereas the turnover of bulk mRNA remains unaffected when SG-formation is impaired. Together these findings indicate that the stabilization of mRNAs during cellular stress is facilitated by the formation of stable mRNPs, which are recruited to SGs by TIA proteins and/or G3BP1. Importantly, however, the aggregation of mRNPs to visible SGs is dispensable for preventing mRNA degradation.     

In silico identification of MicroRNAs targeting the key nucleator of stress granules,G3BP: Promising therapeutics for SARS-CoV-2 infection

Stress granules (SGs) are non-membrane ribonucleoprotein condensates formed in response to environmental stress conditions via liquid–liquid phase separation (LLPS). SGs are involved in the pathogenesis of aging and aging-associated diseases, cancers, viral infection, and several other diseases. GTPase-activating protein (SH3 domain)-binding protein 1 and 2 (G3BP1/2) is a key component and commonly used marker of SGs. Recent studies have shown that SARS-CoV-2 nucleocapsid protein via sequestration of G3BPs inhibits SGs formation in the host cells. In this study, we have identified putative miRNAs targeting G3BP in search of modulators of the G3BP expression. These miRNAs could be considered as new therapeutic targets against COVID-19 infection via the regulation of SG assembly and dynamics.     

G3BP1 promotes stress-induced RNA granule interactions to preserve polyadenylated mRNA

G3BP1, a target of TDP-43, is required for normal stress granule (SG) assembly, but the functional consequences of failed SG assembly remain unknown. Here, using both transformed cell lines and primary neurons, we investigated the functional impact of this disruption in SG dynamics. While stress-induced translational repression and recruitment of key SG proteins was undisturbed, depletion of G3BP1 or its upstream regulator TDP-43 disturbed normal interactions between SGs and processing bodies (PBs). This was concomitant with decreased SG size, reduced SG–PB docking, and impaired preservation of polyadenylated mRNA. Reintroduction of G3BP1 alone was sufficient to rescue all of these phenotypes, indicating that G3BP1 is essential for normal SG–PB interactions and SG function.     

Response to stress in biological disorders: Implications of stress granule assembly and function

It is indispensable for cells to adapt and respond to environmental stresses, in order for organisms to survive. Stress granules (SGs) are condensed membrane‐less organelles dynamically formed in the cytoplasm of eukaryotes cells to cope with diverse intracellular or extracellular stress factors, with features of liquid‐liquid phase separation. They are composed of multiple constituents, including translationally stalled mRNAs, translation initiation factors, RNA‐binding proteins and also non‐RNA‐binding proteins. SG formation is triggered by stress stimuli, viral infection and signal transduction, while aberrant assembly of SGs may contribute to tissue degenerative diseases. Recently, a growing body of evidence has emerged on SG response mechanisms for cells facing high temperatures, oxidative stress and osmotic stress. In this review, we aim to summarize factors affecting SGs assembly, present the impact of SGs on germ cell development and other biological processes. We particularly emphasize the significance of recently reported RNA modifications in SG stress responses. In parallel, we also review all current perspectives on the roles of SGs in male germ cells, with a particular focus on the dynamics of SG assembly.     

Analysis of stress granule assembly in Schizosaccharomyces pombe

Stress granules (SGs) are cytoplasmic aggregates of RNA and proteins in eukaryotic cells that are rapidly induced in response to environmental stress, but are not seen in cells growing under favorable conditions. SGs have been primarily studied in mammalian cells. The existence of SGs in the fission yeast and the distantly related budding yeast was demonstrated only recently. In both species, they contain many orthologs of the proteins seen in mammalian SGs. In this study, we have characterized these proteins and determined their involvement in the assembly of fission yeast SGs, in particular, the homolog of human G3BP proteins. G3BP interacts with the deubiquitinating protease USP10 and plays an important role in the assembly of SGs. We have also identified Ubp3, an ortholog of USP10, as an interaction partner of the fission yeast G3BP-like protein Nxt3 and required for its stability. Under thermal stress, like their human orthologs, both Nxt3 and Ubp3 rapidly relocalize to cytoplasmic foci that contain the SG marker poly(A)-binding protein Pabp. However, in contrast to G3BP1 and USP10, neither deletion nor overexpression of nxt3(+) or ubp3(+) affected the assembly of fission yeast SGs as judged by the relocalization of Pabp. Similar results were observed in mutants defective in orthologs of SG components that are known to affect SG assembly in human and in budding yeast, such as ataxia-2 and TIA-like proteins. Together, our data indicate that despite similar protein compositions, the underlying molecular mechanisms for the assembly of SGs could be distinct between species.     

Inhibition of cytoplasmic mRNA stress granule formation by a viral proteinase

Mammalian cells form dynamic cytoplasmic mRNA stress granules (SGs) in response to environmental stresses including viral infections. SGs are involved in regulating host mRNA function and metabolism, although their precise role during viral infection is unknown. SGs are thought to assemble based on functions of the RNA-binding proteins TIA-1/TIAR or Ras-GAP SH3 domain-binding protein (G3BP). Here, we investigated the relationship between a prototypical plus-strand RNA virus and SGs. Early during poliovirus infection, SG formation is induced, but as infection proceeds this ability is lost, and SGs disperse. Infection resulted in cleavage of G3BP, but not TIA-1 or TIAR, by poliovirus 3C proteinase. Expression of a cleavage-resistant G3BP restored SG formation during poliovirus infection and significantly inhibited virus replication. These results elucidate a mechanism for viral interference with mRNP metabolism and gene regulation and support a critical role of G3BP in SG formation and restriction of virus replication.     

The RasGAP-associated endoribonuclease G3BP assembles stress granules

Stress granules (SGs) are formed in the cytoplasm in response to various toxic agents, and are believed to play a critical role in the regulation of mRNA metabolism during stress. In SGs, mRNAs are stored in an abortive translation initiation complex that can be routed to either translation initiation or degradation. Here, we show that G3BP, a phosphorylation-dependent endoribonuclease that interacts with RasGAP, is recruited to SGs in cells exposed to arsenite. G3BP may thus determine the fate of mRNAs during cellular stress. Remarkably, SG assembly can be either dominantly induced by G3BP overexpression, or on the contrary, inhibited by expressing a central domain of G3BP. This region binds RasGAP and contains serine 149, whose dephosphorylation is induced by arsenite treatment. Critically, a phosphomimetic mutant (S149E) fails to oligomerize and to assemble SGs, whereas a nonphosphorylatable G3BP mutant (S149A) does both. These results suggest that G3BP is an effector of SG assembly, and that Ras signaling contributes to this process by regulating G3BP dephosphorylation.     

The involvement of stress granules in aging and aging‐associated diseases

Stress granules (SGs) are nonmembrane assemblies formed in cells in response to stress conditions. SGs mainly contain untranslated mRNA and a variety of proteins. RNAs and scaffold proteins with intrinsically disordered regions or RNA‐binding domains are essential for the assembly of SGs, and multivalent macromolecular interactions among these components are thought to be the driving forces for SG assembly. The SG assembly process includes regulation through post‐translational modification and involvement of the cytoskeletal system. During aging, many intracellular bioprocesses become disrupted by factors such as cellular environmental changes, mitochondrial dysfunction, and decline in the protein quality control system. Such changes could lead to the formation of aberrant SGs, as well as alterations in their maintenance, disassembly, and clearance. These aberrant SGs might in turn promote aging and aging‐associated diseases. In this paper, we first review the latest progress on the molecular mechanisms underlying SG assembly and SG functioning under stress conditions. Then, we provide a detailed discussion of the relevance of SGs to aging and aging‐associated diseases.     

G3BP–Caprin1–USP10 complexes mediate stress granule condensation and associate with 40S subunits

Mammalian stress granules (SGs) contain stalled translation preinitiation complexes that are assembled into discrete granules by specific RNA-binding proteins such as G3BP. We now show that cells lacking both G3BP1 and G3BP2 cannot form SGs in response to eukaryotic initiation factor 2α phosphorylation or eIF4A inhibition, but are still SG-competent when challenged with severe heat or osmotic stress. Rescue experiments using G3BP1 mutants show that phosphomimetic G3BP1-S149E fails to rescue SG formation, whereas G3BP1-F33W, a mutant unable to bind G3BP partner proteins Caprin1 or USP10, rescues SG formation. Caprin1/USP10 binding to G3BP is mutually exclusive: Caprin binding promotes, but USP10 binding inhibits, SG formation. G3BP interacts with 40S ribosomal subunits through its RGG motif, which is also required for G3BP-mediated SG formation. We propose that G3BP mediates the condensation of SGs by shifting between two different states that are controlled by the phosphorylation of S149 and by binding to Caprin1 or USP10.     

Stress granules contribute to α-globin homeostasis in differentiating erythroid cells

Hemoglobin is the major biosynthetic product of developing erythroid cells. Assembly of hemoglobin requires the balanced production of globin proteins and the oxygen-carrying heme moiety. The heme-regulated inhibitor kinase (HRI) participates in this process by phosphorylating eIF2α and inhibiting the translation of globin proteins when levels of free heme are limiting. HRI is also activated in erythroid cells subjected to oxidative stress. Phospho-eIF2α-mediated translational repression induces the assembly of stress granules (SG), cytoplasmic foci that harbor untranslated mRNAs and promote the survival of cells subjected to adverse environmental conditions. We have found that differentiating erythroid, but not myelomonocytic or megakaryocytic, murine and human progenitor cells assemble SGs, in vitro and in vivo. Targeted knockdown of HRI or G3BP, a protein required for SG assembly, inhibits spontaneous and arsenite-induced assembly of SGs in erythroid progenitor cells. This is accompanied by reduced α-globin production and increased apoptosis suggesting that G3BP+ SGs facilitate the survival of developing erythroid cells.     

RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2 alpha to the assembly of mammalian stress granules

In response to environmental stress, the related RNA-binding proteins TIA-1 and TIAR colocalize with poly(A)(+) RNA at cytoplasmic foci that resemble the stress granules (SGs) that harbor untranslated mRNAs in heat shocked plant cells (Nover et al. 1989; Nover et al. 1983; Scharf et al. 1998). The accumulation of untranslated mRNA at SGs is reversible in cells that recover from a sublethal stress, but irreversible in cells subjected to a lethal stress. We have found that the assembly of TIA-1/R(+) SGs is initiated by the phosphorylation of eIF-2alpha. A phosphomimetic eIF-2alpha mutant (S51D) induces the assembly of SGs, whereas a nonphosphorylatable eIF-2alpha mutant (S51A) prevents the assembly of SGs. The ability of a TIA-1 mutant lacking its RNA-binding domains to function as a transdominant inhibitor of SG formation suggests that this RNA-binding protein acts downstream of the phosphorylation of eIF-2alpha to promote the sequestration of untranslated mRNAs at SGs. The assembly and disassembly of SGs could regulate the duration of stress- induced translational arrest in cells recovering from environmental stress.     

Visibly stressed: the role of eIF2, TIA-1, and stress granules in protein translation

Eukaryotic cells express a family of eukaryotic translation initiation factor 2 alpha (eIF2alpha) kinases (eg, PKR, PERK-PEK, GCN2, HRI) that are individually activated in response to distinct types of environmental stress. Phosphorylation of eIF2alpha by one or more of these kinases reduces the concentration of eIF2-guanosine triphosphate (GTP)-transfer ribonucleic acid for methionine (tRNA(Met)), the ternary complex that loads tRNA(Met) onto the small ribosomal subunit to initiate protein translation. When ternary complex levels are reduced, the related RNA-binding proteins TIA-1 and TIAR promote the assembly of a noncanonical preinitiation complex that lacks eIF2-GTP-tRNA(Met). The TIA proteins dynamically sort these translationally incompetent preinitiation complexes into discrete cytoplasmic domains known as stress granules (SGs). RNA-binding proteins that stabilize or destabilize messenger RNA (mRNA) are also recruited to SGs during stress. Thus, TIA-1 and TIAR act downstream of eIF2alpha phosphorylation to promote SG assembly and facilitate mRNA triage during stress. The role of the SG in the integration of translational efficiency, mRNA stability, and the stress response is discussed.     

Polyamines inhibit the assembly of stress granules in normal intestinal epithelial cells regulating apoptosis

Polyamines regulate multiple signaling pathways and are implicated in many aspects of cellular functions, but the exact molecular processes governed by polyamines remain largely unknown. In response to environmental stress, repression of translation is associated with the assembly of stress granules (SGs) that contain a fraction of arrested mRNAs and are thought to function as mRNA storage. Here we show that polyamines modulate the assembly of SGs in normal intestinal epithelial cells (IECs) and that induced SGs following polyamine depletion are implicated in the protection of IECs against apoptosis. Increasing the levels of cellular polyamines by ectopic overexpression of the ornithine decarboxylase gene decreased cytoplasmic levels of SG-signature constituent proteins eukaryotic initiation factor 3b and T-cell intracellular antigen-1 (TIA-1)-related protein and repressed the assembly of SGs induced by exposure to arsenite-induced oxidative stress. In contrast, depletion of cellular polyamines by inhibiting ornithine decarboxylase with α-difluoromethylornithine increased cytoplasmic eukaryotic initiation factor 3b and TIA-1 related protein abundance and enhanced arsenite-induced SG assembly. Polyamine-deficient cells also exhibited an increase in resistance to tumor necrosis factor-α/cycloheximide-induced apoptosis, which was prevented by inhibiting SG formation with silencing SG resident proteins Sort1 and TIA-1. These results indicate that the elevation of cellular polyamines represses the assembly of SGs in normal IECs and that increased SGs in polyamine-deficient cells are crucial for increased resistance to apoptosis.     

Regulation of AR mRNA translation in response to acute AR pathway inhibition

We report a new mechanism of androgen receptor (AR) mRNA regulation and cytoprotection in response to AR pathway inhibition (ARPI) stress in prostate cancer (PCA). AR mRNA translation is coordinately regulated by RNA binding proteins, YTHDF3 and G3BP1. Under ambient conditions m6A-modified AR mRNA is bound by YTHDF3 and translationally stimulated, while m6A-unmodified AR mRNA is bound by G3BP1 and translationally repressed. When AR-regulated PCA cell lines are subjected to ARPI stress, m6A-modified AR mRNA is recruited from actively translating polysomes (PSs) to RNA-protein stress granules (SGs), leading to reduced AR mRNA translation. After ARPI stress, m6A-modified AR mRNA liquid–liquid phase separated with YTHDF3, while m6A-unmodified AR mRNA phase separated with G3BP1. Accordingly, these AR mRNA messages form two distinct YTHDF3-enriched or G3BP1-enriched clusters in SGs. ARPI-induced SG formation is cell-protective, which when blocked by YTHDF3 or G3BP1 silencing increases PCA cell death in response to ARPI stress. Interestingly, AR mRNA silencing also delays ARPI stress-induced SG formation, highlighting its supportive role in triggering this stress response. Our results define a new mechanism for stress adaptive cell survival after ARPI stress involving SG-regulated translation of AR mRNA, mediated by m6A RNA modification and their respective regulatory proteins.     

Selenite targets eIF4E-binding protein-1 to inhibit translation initiation and induce the assembly of non-canonical stress granules

Stress granules (SGs) are large cytoplasmic ribonucleoprotein complexes that are assembled when cells are exposed to stress. SGs promote the survival of stressed cells by contributing to the reprogramming of protein expression as well as by blocking pro-apoptotic signaling cascades. These cytoprotective effects implicated SGs in the resistance of cancer cells to radiation and chemotherapy. We have found that sodium selenite, a selenium compound with chemotherapeutic potential, is a potent inducer of SG assembly. Selenite-induced SGs differ from canonical mammalian SGs in their morphology, composition and mechanism of assembly. Their assembly is induced primarily by eIF4E-binding protein1 (4EBP1)-mediated inhibition of translation initiation, which is reinforced by concurrent phosphorylation of eIF2α. Selenite-induced SGs lack several classical SG components, including proteins that contribute to pro-survival functions of canonical SGs. Our results reveal a new mechanism of mammalian SG assembly and provide insights into how selenite cytotoxicity may be exploited as an anti-neoplastic therapy.     

Cytoplasmic hGle1A regulates stress granules by modulation of translation

When eukaryotic cells respond to stress, gene expression pathways change to selectively export and translate subsets of mRNAs. Translationally repressed mRNAs accumulate in cytoplasmic foci known as stress granules (SGs). SGs are in dynamic equilibrium with the translational machinery, but mechanisms controlling this are unclear. Gle1 is required for DEAD-box protein function during mRNA export and translation. We document that human Gle1 (hGle1) is a critical regulator of translation during stress. hGle1 is recruited to SGs, and hGLE1 small interfering RNA–mediated knockdown perturbs SG assembly, resulting in increased numbers of smaller SGs. The rate of SG disassembly is also delayed. Furthermore, SG hGle1-depletion defects correlate with translation perturbations, and the hGle1 role in SGs is independent of mRNA export. Interestingly, we observe isoform-specific roles for hGle1 in which SG function requires hGle1A, whereas mRNA export requires hGle1B. We find that the SG defects in hGle1-depleted cells are rescued by puromycin or DDX3 expression. Together with recent links of hGLE1 mutations in amyotrophic lateral sclerosis patients, these results uncover a paradigm for hGle1A modulating the balance between translation and SGs during stress and disease.     

YB-1 regulates stress granule formation and tumor progression by translationally activating G3BP1

Under cell stress, global protein synthesis is inhibited to preserve energy. One mechanism is to sequester and silence mRNAs in ribonucleoprotein complexes known as stress granules (SGs), which contain translationally silent mRNAs, preinitiation factors, and RNA-binding proteins. Y-box binding protein 1 (YB-1) localizes to SGs, but its role in SG biology is unknown. We now report that YB-1 directly binds to and translationally activates the 5′ untranslated region (UTR) of G3BP1 mRNAs, thereby controlling the availability of the G3BP1 SG nucleator for SG assembly. YB-1 inactivation in human sarcoma cells dramatically reduces G3BP1 and SG formation in vitro. YB-1 and G3BP1 expression are highly correlated in human sarcomas, and elevated G3BP1 expression correlates with poor survival. Finally, G3BP1 down-regulation in sarcoma xenografts prevents in vivo SG formation and tumor invasion, and completely blocks lung metastasis in mouse models. Together, these findings demonstrate a critical role for YB-1 in SG formation through translational activation of G3BP1, and highlight novel functions for SGs in tumor progression.