Uncoupling Stress Granule Assembly and Translation Initiation Inhibition

Cytoplasmic stress granules (SGs) are specialized regulatory sites of mRNA translation that form under different stress conditions known to inhibit translation initiation. The formation of SG occurs via two pathways; the eukaryotic initiation factor (eIF) 2α phosphorylation-dependent pathway mediated by stress and the eIF2α phosphorylation-independent pathway mediated by inactivation of the translation initiation factors eIF4A and eIF4G. In this study, we investigated the effects of targeting different translation initiation factors and steps in SG formation in HeLa cells. By depleting eIF2α, we demonstrate that reduced levels of the eIF2.GTP.Met-tRNAi^Met ternary translation initiation complexes is sufficient to induce SGs. Likewise, reduced levels of eIF4B, eIF4H, or polyA-binding protein, also trigger SG formation. In contrast, depletion of the cap-binding protein eIF4E or preventing its assembly into eIF4F results in modest SG formation. Intriguingly, interfering with the last step of translation initiation by blocking the recruitment of 60S ribosome either with 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamideis or through depletion of the large ribosomal subunits protein L28 does not induce SG assembly. Our study identifies translation initiation steps and factors involved in SG formation as well as those that can be targeted without induction of SGs.     

Sequestration of TRAF2 into stress granules interrupts tumor necrosis factor signaling under stress conditions

The cellular stress response (SR) is a phylogenetically conserved protection mechanism that involves inhibition of protein synthesis through recruitment of translation factors such as eIF4G into insoluble stress granules (SGs) and blockade of proinflammatory responses by interruption of the signaling pathway from tumor necrosis factor alpha (TNF-alpha) to nuclear factor-kappaB (NF-kappaB) activation. However, the link between these two physiological phenomena has not been clearly elucidated. Here we report that eIF4GI, which is a scaffold protein interacting with many translation factors, interacts with TRAF2, a signaling molecule that plays a key role in activation of NF-kappaB through TNF-alpha. These two proteins colocalize in SGs during cellular exposure to stress conditions. Moreover, TRAF2 is absent from TNFR1 complexes under stress conditions even after TNF-alpha treatment. This suggests that stressed cells lower their biological activities by sequestration of translation factors and TRAF2 into SGs through a protein-protein interaction.     

Cellular acidosis inhibits assembly, disassembly, and motility of stress granules

Stress granules (SGs) are large ribonucleoprotein (RNP)-containing particles that form in cytoplasm in response to a variety of acute changes in the cellular environment. One of the general parameters of the cell environment is pH. In some diseases, as well as in muscle fatigue, tissue acidosis occurs, leading to decrease in intracellular pH. Here we studied whether decrease in pH causes the formation of SGs in cultured animal cells, whether it affects the formation of the SGs under the action of arsenite and, if such effects occur, what are the mechanisms of the influence of acidosis. Acidosis was simulated by decreasing the pH of the culture medium, which acidified the cytoplasm. We found that medium acidification to pH 6.0 in itself did not cause formation of SGs in cells. Moreover, acidification prevented the formation of SGs under treatment with sodium arsenite or sodium arsenite together with the proteasome inhibitor MG132, and it inhibited the dissociation of preformed SGs under the influence of cycloheximide. We established that pH decrease did not affect the phosphorylation of eIF2α that occurs under the action of sodium arsenite, and even caused such phosphorylation by itself. We also found that the velocity of SG motion in cytoplasm at acidic pH was very low, and the mobile fraction of SG-incorporated PABP protein revealed by FRAP was decreased. We suppose that acidic pH impairs biochemical processes favoring assembly of RNPs in stress conditions and RNP dissociation on the termination of stress. Thus, in acidosis the reaction of the cellular translation apparatus to stress is modified.     

Stress granule assembly is mediated by prion-like aggregation of TIA-1

TIA-1 is an RNA binding protein that promotes the assembly of stress granules (SGs), discrete cytoplasmic inclusions into which stalled translation initiation complexes are dynamically recruited in cells subjected to environmental stress. The RNA recognition motifs of TIA-1 are linked to a glutamine-rich prion-related domain (PRD). Truncation mutants lacking the PRD domain do not induce spontaneous SGs and are not recruited to arsenite-induced SGs, whereas the PRD forms aggregates that are recruited to SGs in low-level-expressing cells but prevent SG assembly in high-level-expressing cells. The PRD of TIA-1 exhibits many characteristics of prions: concentration-dependent aggregation that is inhibited by the molecular chaperone heat shock protein (HSP)70; resistance to protease digestion; sequestration of HSP27, HSP40, and HSP70; and induction of HSP70, a feedback regulator of PRD disaggregation. Substitution of the PRD with the aggregation domain of a yeast prion, SUP35-NM, reconstitutes SG assembly, confirming that a prion domain can mediate the assembly of SGs. Mouse embryomic fibroblasts (MEFs) lacking TIA-1 exhibit impaired ability to form SGs, although they exhibit normal phosphorylation of eukaryotic initiation factor (eIF)2alpha in response to arsenite. Our results reveal that prion-like aggregation of TIA-1 regulates SG formation downstream of eIF2alpha phosphorylation in response to stress.     

Evidence That Ternary Complex (eIF2-GTP-tRNAiMet)–Deficient Preinitiation Complexes Are Core Constituents of Mammalian Stress Granules

Environmental stress-induced phosphorylation of eIF2alpha inhibits protein translation by reducing the availability of eIF2-GTP-tRNA(i)Met, the ternary complex that joins initiator tRNA(Met) to the 43S preinitiation complex. The resulting untranslated mRNA is dynamically routed to discrete cytoplasmic foci known as stress granules (SGs), a process requiring the related RNA-binding proteins TIA-1 and TIAR. SGs appear to be in equilibrium with polysomes, but the nature of this relationship is obscure. We now show that most components of the 48S preinitiation complex (i.e., small, but not large, ribosomal subunits, eIF3, eIF4E, eIF4G) are coordinately recruited to SGs in arsenite-stressed cells. In contrast, eIF2 is not a component of newly assembled SGs. Cells expressing a phosphomimetic mutant (S51D) of eIF2alpha assemble SGs of similar composition, confirming that the recruitment of these factors is a direct consequence of blocked translational initiation and not due to other effects of arsenite. Surprisingly, phospho-eIF2alpha is recruited to SGs that are disassembling in cells recovering from arsenite-induced stress. We discuss these results in the context of a translational checkpoint model wherein TIA and eIF2 are functional antagonists of translational initiation, and in which lack of ternary complex drives SG assembly.     

Critical roles of RNA helicase DDX3 and its interactions with eIF4E/PABP1 in stress granule assembly and stress response

Upon environmental insults, SGs (stress granules) aid cell survival by serving as sites of translational silencing. RNA helicase DDX3 was reported to associate with SGs. However, its role in SG physiology remains undefined. We have demonstrated previously that DDX3 acts as an eIF4E (eukaryotic initiation factor 4E)-inhibitory protein to suppress translation. In the present study, we indentified the SG marker PABP1 [poly(A)-binding protein 1] as another direct interaction partner of DDX3. We established various stimuli as novel stressors that direct DDX3 with eIF4E and PABP1 into SGs, but not to processing bodies. Interestingly, down-regulation of DDX3 interfered with SG assembly, led to nuclear accumulation of PABP1 and reduced cell viability following stress. Conversely, supplementation with a shRNA (short hairpin RNA)-resistant DDX3 restored SG formation, the translocation of PABP1 into SGs and cell survival. Notably, the SG-inducing capacity of DDX3 is independent of its ATPase and helicase activities, but mapped to the eIF4E-binding region. Moreover, the eIF4E-binding-defective mutant DDX3 was impaired in its SG-inducing ability and protective effect on cell survival under adverse conditions. All together, the present study has characterized DDX3 as a pivotal SG-nucleating factor and illustrates co-ordinative roles for DDX3, eIF4E and PABP1 in integrating environmental stress with translational regulation.     

Hydrogen peroxide induces stress granule formation independent of eIF2α phosphorylation

In cells exposed to environmental stress, inhibition of translation initiation conserves energy for the repair of cellular damage. Untranslated mRNAs that accumulate in these cells move to discrete cytoplasmic foci known as stress granules (SGs). The assembly of SGs helps cells to survive under adverse environmental conditions. We have analyzed the mechanism by which hydrogen peroxide (H(2)O(2))-induced oxidative stress inhibits translation initiation and induces SG assembly in mammalian cells. Our data indicate that H(2)O(2) inhibits translation and induces the assembly of SGs. The assembly of H(2)O(2)-induced SGs is independent of the phosphorylation of eIF2α, a major trigger of SG assembly, but requires remodeling of the cap-binding eIF4F complex. Moreover, H(2)O(2)-induced SGs are compositionally distinct from canonical SGs, and targeted knockdown of eIF4E, a protein required for canonical translation initiation, inhibits H(2)O(2)-induced SG assembly. Our data reveal new aspects of translational regulation induced by oxidative insults.     

Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2α phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2α phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2α to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2α phosphorylation-dependent and -independent pathways that target translation initiation.     

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.     

Mammalian orthoreovirus escape from host translational shutoff correlates with stress granule disruption and is independent of eIF2alpha phosphorylation and PKR

In response to mammalian orthoreovirus (MRV) infection, cells initiate a stress response that includes eIF2α phosphorylation and protein synthesis inhibition. We have previously shown that early in infection, MRV activation of eIF2α phosphorylation results in the formation of cellular stress granules (SGs). In this work, we show that as infection proceeds, MRV disrupts SGs despite sustained levels of phosphorylated eIF2α and, further, interferes with the induction of SGs by other stress inducers. MRV interference with SG formation occurs downstream of eIF2α phosphorylation, suggesting the virus uncouples the cellular stress signaling machinery from SG formation. We additionally examined mRNA translation in the presence of SGs induced by eIF2α phosphorylation-dependent and -independent mechanisms. We found that irrespective of eIF2α phosphorylation status, the presence of SGs in cells correlated with inhibition of viral and cellular translation. In contrast, MRV disruption of SGs correlated with the release of viral mRNAs from translational inhibition, even in the presence of phosphorylated eIF2α. Viral mRNAs were also translated in the presence of phosphorylated eIF2α in PKR(-/-) cells. These results suggest that MRV escape from host cell translational shutoff correlates with virus-induced SG disruption and occurs in the presence of phosphorylated eIF2α in a PKR-independent manner.     

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.     

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.     

A Highlights from MBoC Selection: Translation suppression promotes stress granule formation and cell survival in response to cold shock

Cells respond to different types of stress by inhibition of protein synthesis and subsequent assembly of stress granules (SGs), cytoplasmic aggregates that contain stalled translation preinitiation complexes. Global translation is regulated through the translation initiation factor eukaryotic initiation factor 2α (eIF2α) and the mTOR pathway. Here we identify cold shock as a novel trigger of SG assembly in yeast and mammals. Whereas cold shock–induced SGs take hours to form, they dissolve within minutes when cells are returned to optimal growth temperatures. Cold shock causes eIF2α phosphorylation through the kinase PERK in mammalian cells, yet this pathway is not alone responsible for translation arrest and SG formation. In addition, cold shock leads to reduced mitochondrial function, energy depletion, concomitant activation of AMP-activated protein kinase (AMPK), and inhibition of mTOR signaling. Compound C, a pharmacological inhibitor of AMPK, prevents the formation of SGs and strongly reduces cellular survival in a translation-dependent manner. Our results demonstrate that cells actively suppress protein synthesis by parallel pathways, which induce SG formation and ensure cellular survival during hypothermia.     

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.     

Stress Granules Inhibit Apoptosis by Reducing Reactive Oxygen Species Production

Cells can undergo two alternative fates following exposure to environmental stress: they either induce apoptosis or inhibit apoptosis and then repair the stress-induced alterations. These processes minimize cell loss and prevent the survival of cells with aberrant DNA and protein alterations. These two alternative fates are partly controlled by stress granules (SGs). While arsenite, hypoxia, and heat shock induce the formation of SGs that inhibit apoptosis, X-ray irradiation and genotoxic drugs do not induce SGs, and they are more prone to trigger apoptosis. However, it is unclear precisely how SGs control apoptosis. This study found that SGs suppress the elevation of reactive oxygen species (ROS), and this suppression is essential for inhibiting ROS-dependent apoptosis. This antioxidant activity of SGs is controlled by two SG components, GTPase-activating protein SH3 domain binding protein 1 (G3BP1) and ubiquitin-specific protease 10 (USP10). G3BP1 elevates the steady-state ROS level by inhibiting the antioxidant activity of USP10. However, following exposure to arsenite, G3BP1 and USP10 induce the formation of SGs, which uncovers the antioxidant activity of USP10. We also found that the antioxidant activity of USP10 requires the protein kinase activity of ataxia telangiectasia mutated (ATM). This work reveals that SGs are critical redox regulators that control cell fate under stress conditions.     

Formation of stress granules inhibits apoptosis by suppressing stress-responsive MAPK pathways

When confronted with environmental stress, cells either activate defence mechanisms to survive, or initiate apoptosis, depending on the type of stress. Certain types of stress, such as hypoxia, heatshock and arsenite (type 1 stress), induce cells to assemble cytoplasmic stress granules (SGs), a major adaptive defence mechanism. SGs are multimolecular aggregates of stalled translation pre-initiation complexes that prevent the accumulation of mis-folded proteins. Type 2 stress, which includes X-rays and genotoxic drugs, induce apoptosis through the stress-activated p38 and JNK MAPK (SAPK) pathways. A functional relationship between the SG and SAPK responses is unknown. Here, we report that SG formation negatively regulates the SAPK apoptotic response, and that the signalling scaffold protein RACK1 functions as a mediator between the two responses. RACK1 binds to the stress-responsive MTK1 MAPKKK and facilitates its activation by type 2 stress; however, under conditions of type 1 stress, RACK1 is sequestered into SGs. Thus, type 1 conditions suppress activation of the MTK1-SAPK pathway and apoptosis induced by type 2 stress. These findings may be relevant to the problem of hypoxia-induced resistance to cancer chemotherapy.     

Anti-inflammatory lipid mediator 15d-PGJ2 inhibits translation through inactivation of eIF4A

The signaling lipid molecule 15-deoxy-delta 12,14-prostaglandin J2 (15d-PGJ2) has multiple cellular functions, including anti-inflammatory and antineoplastic activities. Here, we report that 15d-PGJ2 blocks translation through inactivation of translational initiation factor eIF4A. Binding of 15d-PGJ2 to eIF4A blocks the interaction between eIF4A and eIF4G that is essential for translation of many mRNAs. Cysteine 264 in eIF4A is the target site of 15d-PGJ2. The antineoplastic activity of 15d-PGJ2 is likely attributed to inhibition of translation. Moreover, inhibition of translation by 15d-PGJ2 results in stress granule (SG) formation, into which TRAF2 is sequestered. The sequestration of TRAF2 contributes to the anti-inflammatory activity of 15d-PGJ2. These findings reveal a novel cross-talk between translation and inflammatory response, and offer new approaches to develop anticancer and anti-inflammatory drugs that target translation factors including eIF4A.     

Monocyte chemotactic protein-induced protein 1 (MCPIP1) suppresses stress granule formation and determines apoptosis under stress

It is unclear how stress granule (SG) formation and cellular apoptosis are coordinately regulated. MCPIP1 (monocyte chemotactic protein-induced protein 1), also known as Zc3h12a, is a critical regulator of the inflammatory response and immune homeostasis. However, the role of MCPIP1 in stress response remains unknown. Here, we report that overexpression of MCPIP1 inhibited the assembly of SGs in response to various stresses. Conversely, MCPIP1-deficient splenocytes developed more SGs even without stress. On the other hand, overexpression of MCPIP1 sensitized RAW 264.7 cells to apoptosis under stress, whereas MCPIP1-deficient cells were resistant to stress-induced apoptosis. Mutagenesis study showed that the ability of MCPIP1 to repress SG formation is dependent on its deubiquitinating activity. Consistently, MCPIP1 negatively regulated stress-induced phosphorylation of eIF2α and thus released stress-induced inhibition of protein translation. However, MCPIP1 also inhibited 15-deoxy-Δ(12,14)-prostaglandin J(2)-induced SG formation, which was reported to be independent of eIF2α phosphorylation. Taken together, these results suggest that MCPIP1 coordinates SG formation and apoptosis during cellular stress and may play a critical role in immune homeostasis and resolution of macrophage inflammation.     

Encephalomyocarditis Virus Disrupts Stress Granules,the Critical Platform for Triggering Antiviral Innate Immune Responses

In response to stress, cells induce ribonucleoprotein aggregates, termed stress granules (SGs). SGs are transient loci containing translation-stalled mRNA, which is eventually degraded or recycled for translation. Infection of some viruses, including influenza A virus with a deletion of nonstructural protein 1 (IAVΔNS1), induces SG-like protein aggregates. Previously, we showed that IAVΔNS1-induced SGs are required for efficient induction of type I interferon (IFN). Here, we investigated SG formation by different viruses using green fluorescent protein (GFP)-tagged Ras-Gap SH3 domain binding protein 1 (GFP-G3BP1) as an SG probe. HeLa cells stably expressing GFP-G3BP1 were infected with different viruses, and GFP fluorescence was monitored live with time-lapse microscopy. SG formations by different viruses was classified into 4 different patterns: no SG formation, stable SG formation, transient SG formation, and alternate SG formation. We focused on encephalomyocarditis virus (EMCV) infection, which exhibited transient SG formation. We found that EMCV disrupts SGs by cleavage of G3BP1 at late stages of infection (>8 h) through a mechanism similar to that used by poliovirus. Expression of a G3BP1 mutant that is resistant to the cleavage conferred persistent formation of SGs as well as an enhanced induction of IFN and other cytokines at late stages of infection. Additionally, knockdown of endogenous G3BP1 blocked SG formation with an attenuated induction of IFN and potentiated viral replication. Taken together, our findings suggest a critical role of SGs as an antiviral platform and shed light on one of the mechanisms by which a virus interferes with host stress and subsequent antiviral responses.