The stress granule protein Vgl1 and poly(A)-binding protein Pab1 are required for doxorubicin resistance in the fission yeast Schizosaccharomyces pombe

Doxorubicin is an anthracycline antibiotic widely used for chemotherapy. Although doxorubicin is effective in the treatment of several cancers, including solid tumors and leukemias, the basis of its mechanism of action is not completely understood. Here, we describe the effects of doxorubicin and its relationship with stress granules formation in the fission yeast, Schizosaccharomyces pombe. We show that disruption of genes encoding the components of stress granules, including vgl1(+), which encodes a multi-KH type RNA-binding protein, and pab1(+), which encodes a poly(A)-binding protein, resulted in greater sensitivity to doxorubicin than seen in wild-type cells. Disruption of the vgl1(+) and pab1(+) genes did not confer sensitivity to other anti-cancer drugs such as cisplatin, 5-fluorouracil, and paclitaxel. We also showed that doxorubicin treatment promoted stress granule formation when combined with heat shock. Notably, doxorubicin treatment did not induce hyperphosphorylation of eIF2α, suggesting that doxorubicin is involved in stress granule assembly independent of eIF2α phosphorylation. Our results demonstrate the usefulness of fission yeast for elucidating the molecular targets of doxorubicin toxicity and suggest a novel drug-resistance mechanism involving stress granule assembly.     

Influenza A Virus Host Shutoff Disables Antiviral Stress-Induced Translation Arrest

Influenza A virus (IAV) polymerase complexes function in the nucleus of infected cells, generating mRNAs that bear 5′ caps and poly(A) tails, and which are exported to the cytoplasm and translated by host machinery. Host antiviral defences include mechanisms that detect the stress of virus infection and arrest cap-dependent mRNA translation, which normally results in the formation of cytoplasmic aggregates of translationally stalled mRNA-protein complexes known as stress granules (SGs). It remains unclear how IAV ensures preferential translation of viral gene products while evading stress-induced translation arrest. Here, we demonstrate that at early stages of infection both viral and host mRNAs are sensitive to drug-induced translation arrest and SG formation. By contrast, at later stages of infection, IAV becomes partially resistant to stress-induced translation arrest, thereby maintaining ongoing translation of viral gene products. To this end, the virus deploys multiple proteins that block stress-induced SG formation: 1) non-structural protein 1 (NS1) inactivates the antiviral double-stranded RNA (dsRNA)-activated kinase PKR, thereby preventing eIF2α phosphorylation and SG formation; 2) nucleoprotein (NP) inhibits SG formation without affecting eIF2α phosphorylation; 3) host-shutoff protein polymerase-acidic protein-X (PA-X) strongly inhibits SG formation concomitant with dramatic depletion of cytoplasmic poly(A) RNA and nuclear accumulation of poly(A)-binding protein. Recombinant viruses with disrupted PA-X host shutoff function fail to effectively inhibit stress-induced SG formation. The existence of three distinct mechanisms of IAV-mediated SG blockade reveals the magnitude of the threat of stress-induced translation arrest during viral replication.     

Formation of antiviral cytoplasmic granules during orthopoxvirus infection

Vaccinia virus (VV) mutants lacking the double-stranded RNA (dsRNA)-binding E3L protein (ΔE3L mutant VV) show restricted replication in most cell types, as dsRNA produced by VV activates protein kinase R (PKR), leading to eIF2α phosphorylation and impaired translation initiation. Here we show that cells infected with ΔE3L mutant VV assemble cytoplasmic granular structures which surround the VV replication factories at an early stage of the nonproductive infection. These structures contain the stress granule-associated proteins G3BP, TIA-1, and USP10, as well as poly(A)-containing RNA. These structures lack large ribosomal subunit proteins, suggesting that they are translationally inactive. Formation of these punctate structures correlates with restricted replication, as they occur in >80% of cells infected with ΔE3L mutant VV but in only 10% of cells infected with wild-type VV. We therefore refer to these structures as antiviral granules (AVGs). Formation of AVGs requires PKR and phosphorylated eIF2α, as mouse embryonic fibroblasts (MEFs) lacking PKR displayed reduced granule formation and MEFs lacking phosphorylatable eIF2α showed no granule formation. In both cases, these decreased levels of AVG formation correlated with increased ΔE3L mutant VV replication. Surprisingly, MEFs lacking the AVG component protein TIA-1 supported increased replication of ΔE3L mutant VV, despite increased eIF2α phosphorylation and the assembly of AVGs that lacked TIA-1. These data indicate that the effective PKR-mediated restriction of ΔE3L mutant VV replication requires AVG formation subsequent to eIF2α phosphorylation. This is a novel finding that supports the hypothesis that the formation of subcellular protein aggregates is an important component of the successful cellular antiviral response.     

Large G3BP-induced granules trigger eIF2α phosphorylation

Stress granules are large messenger ribonucleoprotein (mRNP) aggregates composed of translation initiation factors and mRNAs that appear when the cell encounters various stressors. Current dogma indicates that stress granules function as inert storage depots for translationally silenced mRNPs until the cell signals for renewed translation and stress granule disassembly. We used RasGAP SH3-binding protein (G3BP) overexpression to induce stress granules and study their assembly process and signaling to the translation apparatus. We found that assembly of large G3BP-induced stress granules, but not small granules, precedes phosphorylation of eIF2α. Using mouse embryonic fibroblasts depleted for individual eukaryotic initiation factor 2α (eIF2α) kinases, we identified protein kinase R as the principal kinase that mediates eIF2α phosphorylation by large G3BP-induced granules. These data indicate that increasing stress granule size is associated with a threshold or switch that must be triggered in order for eIF2α phosphorylation and subsequent translational repression to occur. Furthermore, these data suggest that stress granules are active in signaling to the translational machinery and may be important regulators of the innate immune response.     

Myosin Va is required for P body but not stress granule formation

In the present study we demonstrate an association between mammalian myosin Va and cytoplasmic P bodies, microscopic ribonucleoprotein granules that contain components of the 5'-3' mRNA degradation machinery. Myosin Va colocalizes with several P body markers and its RNAi-mediated knockdown results in the disassembly of P bodies. Overexpression of a dominant-negative mutant of myosin Va reduced the motility of P bodies in living cells. Co-immunoprecipitation experiments demonstrate that myosin Va physically associates with eIF4E, an mRNA binding protein that localizes to P bodies. In contrast, we find that myosin Va does not play a role in stress granule formation. Stress granules are ribonucleoprotein structures that are involved in translational silencing and are spatially, functionally, and compositionally linked to P bodies. Myosin Va is found adjacent to stress granules in stressed cells but displays minimal localization within stress granules, and myosin Va knockdown has no effect on stress granule assembly or disassembly. Combined with recently published reports demonstrating a role for Drosophila and mammalian class V myosins in mRNA transport and the involvement of the yeast myosin V orthologue Myo2p in P body assembly, our results provide further evidence that the class V myosins serve an important role in the transport and turnover of mRNA.     

P bodies promote stress granule assembly in Saccharomyces cerevisiae

Recent results indicate that nontranslating mRNAs in eukaryotic cells exist in distinct biochemical states that accumulate in P bodies and stress granules, although the nature of interactions between these particles is unknown. We demonstrate in Saccharomyces cerevisiae that RNA granules with similar protein composition and assembly mechanisms as mammalian stress granules form during glucose deprivation. Stress granule assembly is dependent on P-body formation, whereas P-body assembly is independent of stress granule formation. This suggests that stress granules primarily form from mRNPs in preexisting P bodies, which is also supported by the kinetics of P-body and stress granule formation both in yeast and mammalian cells. These observations argue that P bodies are important sites for decisions of mRNA fate and that stress granules, at least in yeast, primarily represent pools of mRNAs stalled in the process of reentry into translation from P bodies.     

Disruption of microtubules inhibits cytoplasmic ribonucleoprotein stress granule formation

Stress granules are RNP-containing particles arising in the cytoplasm in response to environmental stress. They are dynamic structures assembling and disassembling in the cytoplasm very rapidly. We have studied whether the cytoskeleton is involved in the formation of stress granules. Stress granules were induced in CV-1 cells by sodium arsenate treatment and visualized by immunofluorescent staining with antibodies either to the p170 subunit of eIF3 or to poly(A)-binding protein. Treatment with sodium arsenate for 30-120 min led to assembling of stress granules in a majority of CV-1 cells. Disruption of MT array with nocodazole treatment abolished arsenate-induced formation of stress granules. A similar effect was induced by the microtubule-depolymerizing drug vinblastine, though the influence of the microtubule-stabilizing drug paclitaxel was opposite. Nocodazole treatment did not prevent arsenate-induced phosphorylation of the eIF-2alpha factor, essential for stress granule formation, suggesting that the presence of intact MT array is required for granule assembly. Unexpectedly, treatment of cells with the actin filament-disrupting drug latrunculin B slightly enhanced stress granule formation. We propose that stress granule formation is microtubule-dependent process and likely is facilitated by the motor protein-driven movement of individual stress granule components (e.g., mRNP) along microtubules.     

eIF5A Promotes Translation Elongation,Polysome Disassembly and Stress Granule Assembly

Stress granules (SGs) are cytoplasmic foci at which untranslated mRNAs accumulate in cells exposed to environmental stress. We have identified ornithine decarboxylase (ODC), an enzyme required for polyamine synthesis, and eIF5A, a polyamine (hypusine)-modified translation factor, as proteins required for arsenite-induced SG assembly. Knockdown of deoxyhypusine synthase (DHS) or treatment with a deoxyhypusine synthase inhibitor (GC7) prevents hypusine modification of eIF5A as well as arsenite-induced polysome disassembly and stress granule assembly. Time-course analysis reveals that this is due to a slowing of stress-induced ribosome run-off in cells lacking hypusine-eIF5A. Whereas eIF5A only marginally affects protein synthesis under normal conditions, it is required for the rapid onset of stress-induced translational repression. Our results reveal that hypusine-eIF5A-facilitated translation elongation promotes arsenite-induced polysome disassembly and stress granule assembly in cells subjected to adverse environmental conditions.     

Vgl1, a multi-KH domain protein,is a novel component of the fission yeast stress granules required for cell survival under thermal stress

Multiple KH-domain proteins, collectively known as vigilins, are evolutionarily highly conserved proteins that are present in eukaryotic organisms from yeast to metazoa. Proposed roles for vigilins include chromosome segregation, messenger RNA (mRNA) metabolism, translation and tRNA transport. As a step toward understanding its biological function, we have identified the fission yeast vigilin, designated Vgl1, and have investigated its role in cellular response to environmental stress. Unlike its counterpart in Saccharomyces cerevisiae, we found no indication that Vgl1 is required for the maintenance of cell ploidy in Schizosaccharomyces pombe. Instead, Vgl1 is required for cell survival under thermal stress, and vgl1Δ mutants lose their viability more rapidly than wild-type cells when incubated at high temperature. As for Scp160 in S. cerevisiae, Vgl1 bound polysomes accumulated at endoplasmic reticulum (ER) but in a microtubule-independent manner. Under thermal stress, Vgl1 is rapidly relocalized from the ER to cytoplasmic foci that are distinct from P-bodies but contain stress granule markers such as poly(A)-binding protein and components of the translation initiation factor eIF3. Together, these observations demonstrated in S. pombe the presence of RNA granules with similar composition as mammalian stress granules and identified Vgl1 as a novel component that required for cell survival under thermal stress.     

An Insect Viral Protein Disrupts Stress Granule Formation in Mammalian Cells

Stress granules (SGs) are cytosolic RNA-protein aggregates assembled during stress-induced translation arrest. Virus infection, in general, modulates and blocks SG formation. We previously showed that the model dicistrovirus Cricket paralysis virus (CrPV) 1A protein blocks stress granule formation in insect cells, which is dependent on a specific arginine 146 residue. CrPV-1A also inhibits SG formation in mammalian cells suggesting that this insect viral protein may be acting on a fundamental process that regulates SG formation. The mechanism underlying this process is not fully understood. Here, we show that overexpression of wild-type CrPV-1A, but not the CrPV-1A(R146A) mutant protein, inhibits distinct SG assembly pathways in HeLa cells. CrPV-1A mediated SG inhibition is independent of the Argonaute-2 (Ago-2) binding domain and the E3 ubiquitin ligase recruitment domain. CrPV-1A expression leads to nuclear poly(A)+ RNA accumulation and is correlated with the localization of CrPV-1A to the nuclear periphery. Finally, we show that the overexpression of CrPV-1A blocks FUS and TDP-43 granules, which are pathological hallmarks of neurodegenerative diseases. We propose a model whereby CrPV-1A expression in mammalian cells blocks SG formation by depleting cytoplasmic mRNA scaffolds via mRNA export inhibition. CrPV-1A provides a new molecular tool to study RNA-protein aggregates and potentially uncouple SG functions.     

Stress granules: RNP-containing cytoplasmic bodies springing up under stress. The structure and mechanism of organization

In this review recent data describing stress granules are summarized. Stress granules are specific RNA-containing structures in the cytoplasm of living cells which arise under stress conditions (e. g. heat shock, UV irradiation, energy depletion and oxidative stress). It became evident that stress granules accumulate non-canonical 48S initiation complexes and contain mRNA with associated proteins, small ribosomal subunits and some initiation factors. Stress granules are depleted with ternary complex and large ribosomal subunit. It's proposed that eIF2alpha phosphorylation and ternary complex decrease can be a trigger for stress granule formation. Shuttling nuclear and cytoplasmic RNA-binding protein TIA-1 plays a crucial role in this process. It's proposed that TIA-1 forms prion-like aggregates, and these aggregates are scaffolds for other components of stress granules. Cytoskeletal structures facilitate the accumulation of stress granule components in local cytoplasmic sites. Investigation of process of stress granule formation is important for understanding of cell reaction to stress and translation regulation mechanisms.     

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.     

Prion protein aggresomes are poly(A)+ ribonucleoprotein complexes that induce a PKR-mediated deficient cell stress response

In mammalian cells, cytoplasmic protein aggregates generally coalesce to form aggresomal particles. Recent studies indicate that prion-infected cells produce prion protein (PrP) aggresomes, and that such aggregates may be present in the brain of infected mice. The molecular activity of PrP aggresomes has not been fully investigated. We report that PrP aggresomes initiate a cell stress response by activating the RNA-dependent protein kinase (PKR). Activated PKR phosphorylates the translation initiation factor eIF2alpha, resulting in protein synthesis shut-off. However, other components of the stress response, including the assembly of poly(A)+ RNA-containing stress granules and the synthesis of heat shock protein 70, are repressed. In situ hybridization experiments and affinity chromatography on oligo(dT)-cellulose showed that PrP aggresomes bind poly(A)+ RNA, and are therefore poly(A)+ ribonucleoprotein complexes. These findings support a model in which PrP aggresomes send neuronal cells into untimely demise by modifying the cell stress response, and by inducing the aggregation of poly(A)+ RNA.     

PKCα binds G3BP2 and regulates stress granule formation following cellular stress

Protein kinase C (PKC) isoforms regulate a number of processes crucial for the fate of a cell. In this study we identify previously unrecognized interaction partners of PKCα and a novel role for PKCα in the regulation of stress granule formation during cellular stress. Three RNA-binding proteins, cytoplasmic poly(A)(+) binding protein (PABPC1), IGF-II mRNA binding protein 3 (IGF2BP3), and RasGAP binding protein 2 (G3BP2) all co-precipitate with PKCα. RNase treatment abolished the association with IGF2BP3 and PABPC1 whereas the PKCα-G3BP2 interaction was largely resistant to this. Furthermore, interactions between recombinant PKCα and G3BP2 indicated that the interaction is direct and PKCα can phosphorylate G3BP2 in vitro. The binding is mediated via the regulatory domain of PKCα and the C-terminal RNA-binding domain of G3BP2. Both proteins relocate to and co-localize in stress granules, but not to P-bodies, when cells are subjected to stress. Heat shock-induced stress granule assembly and phosphorylation of eIF2α are suppressed following downregulation of PKCα by siRNA. In conclusion this study identifies novel interaction partners of PKCα and a novel role for PKCα in regulation of stress granules.     

Mammalian stress granules represent sites of accumulation of stalled translation initiation complexes

In eukaryotic cells subjected to environmental stress, untranslated mRNA accumulates in discrete cytoplasmic foci that have been termed stress granules. Recent studies have shown that in addition to mRNA, stress granules also contain 40S ribosomal subunits and various translation initiation factors, including the mRNA binding proteins eIF4E and eIF4G. However, eIF2, the protein that transfers initiator methionyl-tRNA(i) (Met-tRNA(i)) to the 40S ribosomal subunit, has not been detected in stress granules. This result is surprising because the eIF2. GTP. Met-tRNA(i) complex is thought to bind to the 40S ribosomal subunit before the eIF4G. eIF4E. mRNA complex. In the present study, we show in both NIH-3T3 cells and mouse embryo fibroblasts that stress granules contain not only eIF2 but also the guanine nucleotide exchange factor for eIF2, eIF2B. Moreover, we show that phosphorylation of the alpha-subunit of eIF2 is necessary and sufficient for stress granule formation during the unfolded protein response. Finally, we also show that stress granules contain many, if not all, of the components of the 48S preinitiation complex, but not 60S ribosomal subunits, suggesting that they represent stalled translation initiation complexes.