Component Of Stress Granules Research Articles

Canonical nucleators are dispensable for stress granule assembly in Drosophila intestinal progenitors.

Stressed cells downregulate translation initiation and assemble membrane-less foci termed stress granules (SGs). Although SGs have been extensively characterized in cultured cells, the existence of such structures in stressed adult stem cell pools remains poorly characterized. Here, we report that the Drosophila orthologs of the mammalian SG components AGO1, ATX2, CAPRIN, eIF4E, FMRP, G3BP, LIN-28, PABP and TIAR are enriched in adult fly intestinal progenitor cells, where they accumulate in small cytoplasmic messenger ribonucleoprotein complexes (mRNPs). Treatment with sodium arsenite or rapamycin reorganized these mRNPs into large cytoplasmic granules. Formation of these intestinal progenitor stress granules (IPSGs) depended on polysome disassembly, led to translational downregulation and was reversible. Although the canonical SG nucleators ATX2 and G3BP were sufficient for IPSG formation in the absence of stress, neither of them, nor TIAR, either individually or collectively, were required for stress-induced IPSG formation. This work therefore finds that IPSGs do not assemble via a canonical mechanism, raising the possibility that other stem cell populations employ a similar stress-response mechanism.

In vivo stress granule misprocessing evidenced in a FUS knock-in ALS mouse model.

Many RNA-binding proteins, including TDP-43, FUS, and TIA1, are stress granule components, dysfunction of which causes amyotrophic lateral sclerosis (ALS). However, whether a mutant RNA-binding protein disrupts stress granule processing in vivo in pathogenesis is unknown. Here we establish a FUS ALS mutation, p.R521C, knock-in mouse model that carries impaired motor ability and late-onset motor neuron loss. In disease-susceptible neurons, stress induces mislocalization of mutant FUS into stress granules and upregulation of ubiquitin, two hallmarks of disease pathology. Additionally, stress aggravates motor performance decline in the mutant mouse. By using two-photon imaging in TIA1-EGFP transduced animals, we document more intensely TIA1-EGFP-positive granules formed hours but cleared weeks after stress challenge in neurons in the mutant cortex. Moreover, neurons with severe granule misprocessing die days after stress challenge. Therefore, we argue that stress granule misprocessing is pathogenic in ALS, and the model we provide here is sound for further disease mechanistic study.

DDX3X Cellular Valency Allows Stressed Cells to Make a Live or Die Cell Fate Decision by Regulating Stress Granules and the NLRP3 Inflammasome

Abstract Cells of the immune system have to respond to a variety of stressors to help maintain organismal homeostasis. Stress sensing can lead to the assembly of a cytoplasmic membraneless compartment called stress granules (SGs) that helps the cells survive. Cellular stress can also lead to activation of the NLRP3 inflammasome and induction of pyroptosis – a pro-inflammatory programmed cell death. Thus SGs and the NLRP3 inflammasome provide contrasting cell fate choices to a stressed cell – cell survival or pyroptosis. DDX3X is an integral component of SGs [1]. We show that DDX3X promotes the NLRP3 inflammasome activation and ASC speck assembly[2]. Our data suggests that induction of SGs inhibits the NLRP3 inflammasome activation by sequestering the DDX3X molecules. It also suggests that SGs and the NLRP3 inflammasome compete for DDX3X molecules, which makes DDX3X cellular valency critical for this decision making process[3]. Using LPS mediated peritonitis model, we further show that DDX3X promotes and SGs inhibit the NLRP3 inflammasome activation in vivo. Our data points towards a mechanistic paradigm where cells can exploit the cellular valency of biomolecules and a competition between biological processes for common essential factors to make cell fate decisions.

The Co-Chaperone HspBP1 Is a Novel Component of Stress Granules that Regulates Their Formation.

The co-chaperone HspBP1 interacts with members of the hsp70 family, but also provides chaperone-independent functions. We report here novel biological properties of HspBP1 that are relevant to the formation of cytoplasmic stress granules (SGs). SG assembly is a conserved reaction to environmental or pathological insults and part of the cellular stress response. Our study reveals that HspBP1 (1) is an integral SG constituent, and (2) a regulator of SG assembly. Oxidative stress relocates HspBP1 to SGs, where it co-localizes with granule marker proteins and polyA-RNA. Mass spectrometry and co-immunoprecipitation identified novel HspBP1-binding partners that are critical for SG biology. Specifically, HspBP1 associates with the SG proteins G3BP1, HuR and TIA-1/TIAR. HspBP1 also interacts with polyA-RNA in vivo and binds directly RNA homopolymers in vitro. Multiple lines of evidence and single-granule analyses demonstrate that HspBP1 is crucial for SG biogenesis. Thus, HspBP1 knockdown interferes with stress-induced SG assembly. By contrast, HspBP1 overexpression promotes SG formation in the absence of stress. Notably, the hsp70-binding domains of HspBP1 regulate SG production in unstressed cells. Taken together, we identified novel HspBP1 activities that control SG formation. These features expand HspBP1’s role in the cellular stress response and provide new mechanistic insights into SG biogenesis.

The RNA-Binding Protein Rasputin/G3BP Enhances the Stability and Translation of Its Target mRNAs

G3BP RNA-binding proteins are important components of stress granules (SGs). Here, we analyze the role of the Drosophila G3BP Rasputin (RIN) in unstressed cells, where RIN is not SG associated. Immunoprecipitation followed by microarray analysis identifies over 550 mRNAs that copurify with RIN. The mRNAs found in SGs are long and translationally silent. In contrast, we find that RIN-bound mRNAs, which encode core components of the transcription, splicing, and translation machinery, are short, stable, and highly translated. We show that RIN is associated with polysomes and provide evidence for a direct role for RIN and its human homologs in stabilizing and upregulating the translation of their target mRNAs. We propose that when cells are stressed, the resulting incorporation of RIN/G3BPs into SGs sequesters them away from their short target mRNAs. This would downregulate the expression of these transcripts, even though they are not incorporated into stress granules.

In silico analysis of COSMIC retrieved P body gene mutations in breast cancer

P bodies (Processing bodies) are mRNP granules and are involved in mRNA decay, gene silencing by miRNA or siRNA, deadenylation and decapping. These are dynamic and shuttle their components with stress granules, ribosomes and stalled mRNAs within them can assemble to polysomes. Research suggests that several components of stress granules and P bodies contribute to carcinogenesis. In corroboration, COSMIC (catalogue of somatic mutations in cancer) database documents somatic mutations of P body genes. In our study, we have retrieved the somatic mutations from COSMIC for critical P body genes such as DCP1B, EIF4E, XRN1, MOV10, CNOT6, HTT and GEMIN5 and examined for their deleteriousness using various online tools such as PolyPhen, MutationTaster and STRUM. A total of 5, 2, 17, 11, 8, 22 and 15 breast cancer mutations were retrieved for DCP1B, EIF4E, XRN1, MOV10, CNOT6, HTT and GEMIN5 respectively. Of these, our analysis has identified 18 mutations (DCP1B-1, EIF4E-1, MOV10-1, GEMIN5-2, CNOT6-2, HTT-9 and XRN1-2) to be deleterious. Also, our 3D molecular dynamic studies suggest that HTT and EIF4E mutations induce perturbations in the mutant proteins. P body dynamics are marked by interactions between P body proteins and other cellular components and our results give insights to examine the P body dynamics on such mutation landscapes.

Does Inter-Organellar Proteostasis Impact Yeast Quality and Performance During Beer Fermentation?

During beer production, yeast generate ethanol that is exported to the extracellular environment where it accumulates. Depending on the initial carbohydrate concentration in the wort, the amount of yeast biomass inoculated, the fermentation temperature, and the yeast attenuation capacity, a high concentration of ethanol can be achieved in beer. The increase in ethanol concentration as a consequence of the fermentation of high gravity (HG) or very high gravity (VHG) worts promotes deleterious pleiotropic effects on the yeast cells. Moderate concentrations of ethanol (5% v/v) change the enzymatic kinetics of proteins and affect biological processes, such as the cell cycle and metabolism, impacting the reuse of yeast for subsequent fermentation. However, high concentrations of ethanol (> 5% v/v) dramatically alter protein structure, leading to unfolded proteins as well as amorphous protein aggregates. It is noteworthy that the effects of elevated ethanol concentrations generated during beer fermentation resemble those of heat shock stress, with similar responses observed in both situations, such as the activation of proteostasis and protein quality control mechanisms in different cell compartments, including endoplasmic reticulum (ER), mitochondria, and cytosol. Despite the extensive published molecular and biochemical data regarding the roles of proteostasis in different organelles of yeast cells, little is known about how this mechanism impacts beer fermentation and how different proteostasis mechanisms found in ER, mitochondria, and cytosol communicate with each other during ethanol/fermentative stress. Supporting this integrative view, transcriptome data analysis was applied using publicly available information for a lager yeast strain grown under beer production conditions. The transcriptome data indicated upregulation of genes that encode chaperones, co-chaperones, unfolded protein response elements in ER and mitochondria, ubiquitin ligases, proteasome components, N-glycosylation quality control pathway proteins, and components of processing bodies (p-bodies) and stress granules (SGs) during lager beer fermentation. Thus, the main purpose of this hypothesis and theory manuscript is to provide a concise picture of how inter-organellar proteostasis mechanisms are connected with one another and with biological processes that may modulate the viability and/or vitality of yeast populations during HG/VHG beer fermentation and serial repitching.

HDAC6, A Novel Cargo for Autophagic Clearance of Stress Granules, Mediates the Repression of the Type I Interferon Response During Coxsackievirus A16 Infection.

Autophagic cargoes ensure selective autophagy for the recognition and removal of various cytosolic aggregated proteins, damaged organelles, or pathogens. Stress granules (SGs), as antiviral immune complexes, serve a positive role in the type I interferon (IFN) response and can be targeted by autophagy (termed granulophagy). However, the cargo of granulophagy remains elusive, and it is still unknown whether granulophagy plays a role in viral infection. Here, we found that histone deacetylase 6 (HDAC6), a component of viral RNA-induced SGs, is a novel granulophagic cargo that is recognized by p62/Sequestosome 1 (SQSTM1) and mediates the degradation of SGs in coxsackievirus A16 (CA16)-infected cells. CA16 viral RNA activated the protein kinase RNA-activated (PKR)/eukaryotic translation initiation factor 2-alpha (eIF2α) pathway to promote SG assembly. The SGs were degraded by CA16-triggered autophagy via the interaction between the ubiquitin-associated (UBA) domain of p62 and the ubiquitin-binding domain (UBD) of HDAC6, which was bridged by a poly-ubiquitin chain. We also found that granulophagy repressed the type I interferon response and facilitated viral replication. These results suggest that HDAC6 might be the first identified granulophagic cargo and granulophagy could be a strategy that viruses apply to repress the antiviral immune response.

Drought Stress Causes Specific Changes to the Spliceosome and Stress Granule Components

The spliceosome processes RNAs from a pre-RNA state to a mature mRNA thereby influencing RNA availability for translation, localization, and turnover. It consists of complex structures containing RNA-binding proteins (RBPs) essential for post-transcriptional gene expression control. Here we investigate the dynamic modifications of spliceosomal RBPs under stress and in particular drought stress. We do so by mRNA interactome capture in Arabidopsis thaliana using label free quantitation. This approach identified 44 proteins associated with the spliceosome and further 32 proteins associated with stress granules. We noted a high enrichment in the motifs RDRR and RSRSRS that are characteristic of RNA interacting proteins. Identification of splicing factors reflect direct and/or indirect stress induced splicing events that have a direct effect on transcriptome and proteome changes under stress. Furthermore, detection of stress granule components is consistent with transcriptional arrest. Identification of drought induced stress granule components is critical in determining common abiotic stress-induced foci that can have biotechnological applications. This study may therefore open ways to modify plant stress responses at a systems level through the modification of key spliceosome components.

UBAP2L Forms Distinct Cores that Act in Nucleating Stress Granules Upstream of G3BP1.

Stress granules (SGs) are membraneless organelles that form in eukaryotic cells after stress exposure [1] (reviewed in [2-4]). Following translation inhibition, polysome disassembly releases 48S preinitiation complexes (PICs). mRNA, PICs, and other proteins coalesce in SG cores [1, 5-7]. SG cores recruit a dynamic shell, whose properties are dominated byweak interactions between proteins and RNAs [8-10]. The structure and assembly of SGs and how different components contribute to their formation are not fully understood. Using super-resolution and expansion microscopy, we find that the SG component UBAP2L [11, 12] and the core protein G3BP1 [5, 11-13] occupy different domains inside SGs. UBAP2L displays typical properties of a core protein, indicating that cores of different compositions coexist inside the same granule. Consistent with a role as a core protein, UBAP2L is required forSG assembly in several stress conditions. Our reverse genetic and cell biology experiments suggest that UBAP2L forms granules independent of G3BP1 and 2 but does not interfere with stress-induced translational inhibition. We propose a model in which UBAP2L is an essential SG nucleator that acts upstream of G3BP1 and 2 and facilitates G3BP1 core formation and SG assembly and growth.

Multiple distinct pathways lead to hyperubiquitylated insoluble TDP-43 protein independent of its translocation into stress granules

Multiple distinct pathways lead to hyperubiquitylated insoluble TDP-43 protein independent of its translocation into stress granules

Stress granule: A promising target for cancer treatment.

Stress granules (SGs) are primarily composed of mRNAs that stall at translation initiation and usually appear in the cytoplasm under unusual physiological or pathological conditions such as hypoxia, oxidative stress, and viral infection. Recent studies have indicated that several components of SGs participate in tumourigenesis and cancer metastasis through tumour-associated signalling pathways as well as other mechanisms. Furthermore, some chemotherapy drugs have been reported to induce SGs. Thus, the roles of SGs in cancer treatment have attracted considerable interest. Importantly, disturbing the recruitment of SGs components or microtubule polymerization, as well as other strategies that can abolish SGs formation, is reported to inhibit tumour progression, suggesting that targeting SGs could be a promising strategy for cancer treatment. In this review, we summarize the relationship between SGs and cancer, as well as recent advances in targeting SGs, in the interest of providing new opportunities for cancer treatment.

Properties of Stress Granule and P-Body Proteomes.

Stress granules and P-bodies are cytosolic biomolecular condensates that dynamically form by the phase separation of RNAs and proteins. They participate in translational control and buffer the proteome. Upon stress, global translation halts and mRNAs bound to the translational machinery and other proteins coalesce to form stress granules (SGs). Similarly, translationally stalled mRNAs devoid of translation initiation factors shuttle to P-bodies (PBs). Here, we review the cumulative progress made in defining the protein components that associate with mammalian SGs and PBs. We discuss the composition of SG and PB proteomes, supported by a new user-friendly database (http://rnagranuledb.lunenfeld.ca/) that curates current literature evidence for genes or proteins associated with SGs or PBs. As previously observed, the SG and PB proteomes are biased toward intrinsically disordered regions and have a high propensity to contain primary sequence features favoring phase separation. We also provide an outlook on how the various components of SGs and PBs may cooperate to organize and form membraneless organelles.

Nd1-L as a novel stress granules (SGs) component that regulates stress granule dynamics and associates with autophagy components

Nd1-L as a novel stress granules (SGs) component that regulates stress granule dynamics and associates with autophagy components

The TRAPP complex mediates secretion arrest induced by stress granule assembly.

The TRAnsport Protein Particle (TRAPP) complex controls multiple membrane trafficking steps and is strategically positioned to mediate cell adaptation to diverse environmental conditions, including acute stress. We have identified the TRAPP complex as a component of a branch of the integrated stress response that impinges on the early secretory pathway. The TRAPP complex associates with and drives the recruitment of the COPII coat to stress granules (SGs) leading to vesiculation of the Golgi complex and arrest of ER export. The relocation of the TRAPP complex and COPII to SGs only occurs in cycling cells and is CDK1/2-dependent, being driven by the interaction of TRAPP with hnRNPK, a CDK substrate that associates with SGs when phosphorylated. In addition, CDK1/2 inhibition impairs TRAPP complex/COPII relocation to SGs while stabilizing them at ER exit sites. Importantly, the TRAPP complex controls the maturation of SGs. SGs that assemble in TRAPP-depleted cells are smaller and are no longer able to recruit RACK1 and Raptor, two TRAPP-interactive signaling proteins, sensitizing cells to stress-induced apoptosis.

Noroviruses subvert the core stress granule component G3BP1 to promote viral VPg-dependent translation.

Knowledge of the host factors required for norovirus replication has been hindered by the challenges associated with culturing human noroviruses. We have combined proteomic analysis of the viral translation and replication complexes with a CRISPR screen, to identify host factors required for norovirus infection. The core stress granule component G3BP1 was identified as a host factor essential for efficient human and murine norovirus infection, demonstrating a conserved function across the Norovirus genus. Furthermore, we show that G3BP1 functions in the novel paradigm of viral VPg-dependent translation initiation, contributing to the assembly of translation complexes on the VPg-linked viral positive sense RNA genome by facilitating ribosome recruitment. Our data uncovers a novel function for G3BP1 in the life cycle of positive sense RNA viruses and identifies the first host factor with pan-norovirus pro-viral activity.

The prion-like protein kinase Sky1 is required for efficient stress granule disassembly

Stress granules are membraneless protein- and mRNA-rich organelles that form in response to perturbations in environmental conditions. Stress granule formation is reversible, and persistent stress granules have been implicated in a variety of neurodegenerative disorders, including amyotrophic lateral sclerosis. However, characterization of the factors involved in dissolving stress granules is incomplete. Many stress granule proteins contain prion-like domains (PrLDs), some of which have been linked to stress granule formation. Here, we demonstrate that the PrLD-containing yeast protein kinase Sky1 is a stress granule component. Sky1 is recruited to stress granules in part via its PrLD, and Sky1’s kinase activity regulates timely stress granule disassembly during stress recovery. This effect is mediated by phosphorylation of the stress granule component Npl3. Sky1 can compensate for defects in chaperone-mediated stress granule disassembly and vice-versa, demonstrating that cells have multiple overlapping mechanisms for re-solubilizing stress granule components.

Fibril-induced glutamine-/asparagine-rich prions recruit stress granule proteins in mammalian cells.

Prions of lower eukaryotes are self-templating protein aggregates that replicate by converting homotypic proteins into stable, tightly packed beta-sheet-rich protein assemblies. Propagation is mediated by prion domains, low-complexity regions enriched in polar and devoid of charged amino acid residues. In mammals, compositionally similar domains modulate the assembly of dynamic stress granules (SGs) that associate via multivalent weak interactions. Dysregulation of SGs composed of proteins with prion-like domains has been proposed to underlie the formation of pathological inclusions in several neurodegenerative diseases. The events that drive prion-like domains into transient or solid assemblies are not well understood. We studied the interactors of the prototype prion domain NM of Saccharomyces cerevisiae Sup35 in its soluble or fibril-induced prion conformation in the mammalian cytosol. We show that the interactomes of soluble and prionized NM overlap with that of SGs. Prion induction by exogenous seeds does not cause SG assembly, demonstrating that colocalization of aberrant protein inclusions with SG components does not necessarily reveal SGs as initial sites of protein misfolding.

UBAP2L arginine methylation by PRMT1 modulates stress granule assembly

Stress granules (SGs) are discrete assemblies of stalled messenger ribonucleoprotein complexes (mRNPs) that form when eukaryotic cells encounter environmental stress. RNA-binding proteins (RBPs) mediate their condensation by recruiting populations of mRNPs. However, the cellular and molecular mechanisms underlying the role of ubiquitin-associated protein 2-like (UBAP2L) in the regulation of SG dynamics remain elusive. Here, we show that UBAP2L is required for both SG assembly and disassembly. UBAP2L overexpression nucleated SGs under stress-null conditions. The UBAP2L Arg–Gly–Gly (RGG) motif was required for SG competence, and mediated the recruitment of SG components, including mRNPs, RBPs, and ribosomal subunits. The domain of unknown function (DUF) of UBAP2L-mediated interaction with ras GTPase-activating protein-binding protein (G3BP)1/2, and its deletion caused the cytoplasmic–nuclear transport of UBAP2L and G3BP1/2, thereby compromising SG formation. The protein arginine methyltransferase PRMT1 asymmetrically dimethylated UBAP2L by targeting the RGG motif. Increased arginine methylation blocked, whereas its decrease enhanced UBAP2L interactions with SG components, ablating and promoting SG assembly, respectively. These results provide new insights into the mechanisms by which UBAP2L regulates SG dynamics and RNA metabolism.

Rbfox2 dissociation from stress granules suppresses cancer progression

Stress granules (SGs) are stalled translation initiation complexes comprising untranslated mRNAs and RNA-binding proteins (RBPs). RBP fox-1 homolog 2 (Rbfox2), a component of SGs, binds to retinoblastoma 1 (RB1) mRNA, which is closely related to cancer progression; however, the role of Rbfox2 in cancer progression remains largely unknown. In this study, we confirmed that Rbfox2, which is present in the nucleus as a splicing regulator, localizes to the cytoplasm of human colon cancer tissues and that induction of Rbfox2 dissociation from SGs by resveratrol treatment inhibits cancer progression. We also observed that Rbfox2 in SGs inhibited RB1 protein expression and promoted cell cycle progression. Additionally, resveratrol treatment inhibited SG-mediated Rbfox2 localization, further inhibiting RB1 protein expression, and inhibited specific Rbfox2 localization to the cytoplasm in melanoma B16-F10 cells, thereby effectively inhibiting metastasis and tumor growth ability. These results indicate that Rbfox2 dissociation from SGs attenuates cancer progression and offer insight into the mechanism associated with Rbfox2 dissociation, thereby marking Rbfox2 as a potential candidate target for cancer therapy.