Caprin-1 binding to the critical stress granule protein G3BP1 is influenced by pH.

G3BP is the central node within stress-induced protein-RNA interaction networks known as stress granules (SGs). The SG-associated proteins Caprin-1 and USP10 bind mutually exclusively to the NTF2 domain of G3BP1, promoting and inhibiting SG formation, respectively. Herein, we present the crystal structure of G3BP1-NTF2 in complex with a Caprin-1-derived short linear motif (SLiM). Caprin-1 interacts with His-31 and His-62 within a third NTF2-binding site outside those covered by USP10, as confirmed using biochemical and biophysical-binding assays. Nano-differential scanning fluorimetry revealed reduced thermal stability of G3BP1-NTF2 at acidic pH. This destabilization was counterbalanced significantly better by bound USP10 than Caprin-1. The G3BP1/USP10 complex immunoprecipated from human U2OS cells was more resistant to acidic buffer washes than G3BP1/Caprin-1. Acidification of cellular condensates by approximately 0.5 units relative to the cytosol was detected by ratiometric fluorescence analysis of pHluorin2 fused to G3BP1. Cells expressing a Caprin-1/FGDF chimera with higher G3BP1-binding affinity had reduced Caprin-1 levels and slightly reduced condensate sizes. This unexpected finding may suggest that binding of the USP10-derived SLiM to NTF2 reduces the propensity of G3BP1 to enter condensates.

TSC2 Interacts with HDLBP/Vigilin and Regulates Stress Granule Formation.

Tuberous sclerosis complex (TSC) is caused by mutations of either the TSC1 or TSC2 tumor suppressor gene. TSC causes tumors of the brain, heart, kidney, skin and lymphangioleiomyomatosis (LAM). Here we report that the TSC2 protein physically binds to high-density lipoprotein binding protein (HDLBP), also called vigilin, a core stress granule (SG) protein, and that TSC2 localizes to SGs. SGs contain mRNAs and translation initiation complexes, and regulate gene expression by sequestering specific transcripts, thereby serving a cytoprotective role. TSC2 has never before been shown to localize to SGs and knocking down vigilin impacts SG translocation of TSC2. TSC2-deficient cells showed a striking increase in the number of SGs after thermal shock and arsenite treatment relative to Tsc2-expressing cells. Our findings also show that murine kidney lysates from a model of TSC have increased levels of SG components including G3BP1 and Caprin1. G3BP1 and Caprin are elevated in renal angiomyolipomas (a renal tumor common in patients with TSC) compared with control normal kidney. G3BP1 is also elevated in TSC-associated subependymal giant cell astrocytomas. We found that genetic inhibition of G3BP1 inhibits the proliferation of TSC2-deficient cells in vitro. Finally, in a mouse model of TSC, genetic inhibition of SGs suppresses cell growth, suggesting that targeting SGs may have efficacy in the therapy of TSC. IMPLICATIONS: This study demonstrates that TSC2 physically interacts with HDLBP/vigilin, a component of SGs, that TSC2 localizes to SG and that TSC2-deficient cells have more SGs, suggesting that SGs represent a novel therapeutic target in TSC.

Bisphenol A promotes stress granule assembly and modulates the integrated stress response.

Bisphenol-A (BPA) is a ubiquitous precursor of polycarbonate plastics that is found in the blood and serum of >92% of Americans. While BPA has been well documented to act as a weak estrogen receptor (ER) agonist, its effects on cellular stress are unclear. Here, we demonstrate that high-dose BPA causes stress granules (SGs) in human cells. A common estrogen derivative, ß-estradiol, does not trigger SGs, indicating the mechanism of SG induction is not via the ER pathway. We also tested other structurally related environmental contaminants including the common BPA substitutes BPS and BPF, the industrial chemical 4-nonylphenol (4-NP) and structurally related compounds 4-EP and 4-VP, as well as the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D). The variable results from these related compounds suggest that structural homology is not a reliable predictor of the capacity of a compound to cause SGs. Also, we demonstrate that BPA acts primarily through the PERK pathway to generate canonical SGs. Finally, we show that chronic exposure to a low physiologically relevant dose of BPA suppresses SG assembly upon subsequent acute stress. Interestingly, this SG inhibition does not affect phosphorylation of eIF2α or translation inhibition, thus uncoupling the physical assembly of SGs from translational control. Our work identifies additional effects of BPA beyond endocrine disruption that may have consequences for human health.

Molecular mechanisms of stress granule assembly and disassembly.

Stress granules (SGs) are membrane-less ribonucleoprotein (RNP)-based cellular compartments that form in the cytoplasm of a cell upon exposure to various environmental stressors. SGs contain a large set of proteins, as well as mRNAs that have been stalled in translation as a result of stress-induced polysome disassembly. Despite the fact that SGs have been extensively studied for many years, their function is still not clear. They presumably help the cell to cope with the encountered stress, and facilitate the recovery process after stress removal upon which SGs disassemble. Aberrant formation of SGs and impaired SG disassembly majorly contribute to various pathological phenomena in cancer, viral infections, and neurodegeneration. The assembly of SGs is largely driven by liquid-liquid phase separation (LLPS), however, the molecular mechanisms behind that are not fully understood. Recent studies have proposed a novel mechanism for SG formation that involves the interplay of a large interaction network of mRNAs and proteins. Here, we review this novel concept of SG assembly, and discuss the current insights into SG disassembly.

Spatiotemporal Proteomic Analysis of Stress Granule Disassembly Using APEX Reveals Regulation by SUMOylation and Links to ALS Pathogenesis.

Stress granules (SGs) are cytoplasmic assemblies of proteins and non-translating mRNAs. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SGs undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of the SG temporal disassembly sequence using multi-bait APEX proximity proteomics. We discover 109 novel SG proteins and characterize distinct SG substructures. We reveal dozens of disassembly-engaged proteins (DEPs), some of which play functional roles in SG disassembly, including small ubiquitin-like modifier (SUMO) conjugating enzymes. We further demonstrate that SUMOylation regulates SG disassembly and SG formation. Parallel proteomics with amyotrophic lateral sclerosis (ALS)-associated C9ORF72 dipeptides uncovered attenuated DEP recruitment during SG disassembly and impaired SUMOylation. Accordingly, SUMO activity ameliorated C9ORF72-ALS-related neurodegeneration in Drosophila. By dissecting the SG spatiotemporal proteomic landscape, we provide an in-depth resource for future work on SG function and reveal basic and disease-relevant mechanisms of SG disassembly.

Mammalian stress granules and P bodies at a glance.

Stress granules (SGs) and processing bodies (PBs) are membraneless ribonucleoprotein-based cellular compartments that assemble in response to stress. SGs and PBs form through liquid-liquid phase separation that is driven by high local concentrations of key proteins and RNAs, both of which dynamically shuttle between the granules and the cytoplasm. SGs uniquely contain certain translation initiation factors and PBs are uniquely enriched with factors related to mRNA degradation and decay, although recent analyses reveal much broader protein commonality between these granules. Despite detailed knowledge of their composition and dynamics, the function of SGs and PBs remains poorly understood. Both, however, contain mRNAs, implicating their assembly in the regulation of RNA metabolism. SGs may also serve as hubs that rewire signaling events during stress. By contrast, PBs may constitute RNA storage centers, independent of mRNA decay. The aberrant assembly or disassembly of these granules has pathological implications in cancer, viral infection and neurodegeneration. Here, we review the current concepts regarding the formation, composition, dynamics, function and involvement in disease of SGs and PBs.

Competing Protein-RNA Interaction Networks Control Multiphase Intracellular Organization.

Liquid-liquid phase separation (LLPS) mediates formation of membraneless condensates such as those associated with RNA processing, but the rules that dictate their assembly, substructure, and coexistence with other liquid-like compartments remain elusive. Here, we address the biophysical mechanism of this multiphase organization using quantitative reconstitution of cytoplasmic stress granules (SGs) with attached P-bodies in human cells. Protein-interaction networks can be viewed as interconnected complexes (nodes) of RNA-binding domains (RBDs), whose integrated RNA-binding capacity determines whether LLPS occurs upon RNA influx. Surprisingly, both RBD-RNA specificity and disordered segments of key proteins are non-essential, but modulate multiphase condensation. Instead, stoichiometry-dependent competition between protein networks for connecting nodes determines SG and P-body composition and miscibility, while competitive binding of unconnected proteins disengages networks and prevents LLPS. Inspired by patchy colloid theory, we propose a general framework by which competing networks give rise to compositionally specific and tunable condensates, while relative linkage between nodes underlies multiphase organization.

Class I HDAC inhibitors enhance YB-1 acetylation and oxidative stress to block sarcoma metastasis.

Outcomes for metastatic Ewing sarcoma and osteosarcoma are dismal and have not changed for decades. Oxidative stress attenuates melanoma metastasis, and melanoma cells must reduce oxidative stress to metastasize. We explored this in sarcomas by screening for oxidative stress sensitizers, which identified the class I HDAC inhibitor MS-275 as enhancing vulnerability to reactive oxygen species (ROS) in sarcoma cells. Mechanistically, MS-275 inhibits YB-1 deacetylation, decreasing its binding to 5'-UTRs of NFE2L2 encoding the antioxidant factor NRF2, thereby reducing NFE2L2 translation and synthesis of NRF2 to increase cellular ROS. By global acetylomics, MS-275 promotes rapid acetylation of the YB-1 RNA-binding protein at lysine-81, blocking binding and translational activation of NFE2L2, as well as known YB-1 mRNA targets, HIF1A, and the stress granule nucleator, G3BP1. MS-275 dramatically reduces sarcoma metastasis in vivo, but an MS-275-resistant YB-1K81-to-alanine mutant restores metastatic capacity and NRF2, HIF1α, and G3BP1 synthesis in MS-275-treated mice. These studies describe a novel function for MS-275 through enhanced YB-1 acetylation, thus inhibiting YB-1 translational control of key cytoprotective factors and its pro-metastatic activity.

Phosphorylation of G3BP1-S149 does not influence stress granule assembly.

Tourrière et al. (2013. J. Cell Biol. https://doi.org/10.1083/jcb.200212128) reported that G3BP1-S149 dephosphorylation promotes stress granule formation. We show that constructs used to establish this conclusion contain additional mutations causing these phenotypes, and that S149 phosphorylation status does not change upon stress.

Genetic Perturbation of TIA1 Reveals a Physiological Role in Fear Memory.

TIA1 is a prion-related RNA-binding protein whose capacity to form various types of intracellular aggregates has been implicated in neurodegenerative disease. However, its role in normal brain function is poorly understood. Here, we show that TIA1 bidirectionally modulates stress-dependent synaptic plasticity in the hippocampus, a brain region involved in fear memory and olfactory discrimination learning. At the behavioral level, conditioned odor avoidance is potentiated by TIA1 deletion, whereas overexpression of TIA1 in the ventral hippocampus inhibits both contextual fear memory and avoidance. However, the latter genetic manipulations have little impact on other hippocampus-dependent tasks. Transcriptional profiling indicates that TIA1 presides over a large network of immune system genes with modulatory roles in synaptic plasticity and long-term memory. Our results uncover a physiological and partly sex-dependent function for TIA1 in fear memory and may provide molecular insight into stress-related psychiatric conditions, such as post-traumatic stress disorder (PTSD) and anxiety.

Stress Granules and Processing Bodies in Translational Control.

Stress granules (SGs) and processing bodies (PBs) are non-membrane-enclosed RNA granules that dynamically sequester translationally inactive messenger ribonucleoprotein particles (mRNPs) into compartments that are distinct from the surrounding cytoplasm. mRNP remodeling, silencing, and/or storage involves the dynamic partitioning of closed-loop polyadenylated mRNPs into SGs, or the sequestration of deadenylated, linear mRNPs into PBs. SGs form when stress-activated pathways stall translation initiation but allow elongation and termination to occur normally, resulting in a sudden excess of mRNPs that are spatially condensed into discrete foci by protein:protein, protein:RNA, and RNA:RNA interactions. In contrast, PBs can exist in the absence of stress, when specific factors promote mRNA deadenylation, condensation, and sequestration from the translational machinery. The formation and dissolution of SGs and PBs reflect changes in messenger RNA (mRNA) metabolism and allow cells to modulate the proteome and/or mediate life or death decisions during changing environmental conditions.

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules.

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to stress conditions, are implicated in various aspects of cell metabolism and survival. SGs modulate cellular signaling pathways, post-transcriptional gene expression, and stress response programs. The formation of these mRNA-containing granules is directly connected to cellular translation. SG assembly is triggered by inhibited translation initiation, and SG disassembly is promoted by translation activation or by inhibited translation elongation. This relationship is further highlighted by SG composition. Core SG components are stalled translation pre-initiation complexes, mRNA, and selected RNA-binding Proteins (RBPs). The purpose of SG assembly is to conserve cellular energy by sequestering translationally stalled housekeeping mRNAs, allowing for the enhanced translation of stress-responsive proteins. In addition to the core constituents, such as stalled translation preinitiation complexes, SGs contain a plethora of other proteins and signaling molecules. Defects in SG formation can impair cellular adaptation to stress and can thus promote cell death. SGs and similar RNA-containing granules have been linked to a number of human diseases, including neurodegenerative disorders and cancer, leading to the recent interest in classifying and defining RNA granule subtypes. This protocol describes assays to characterize and quantify mammalian SGs.

Phase Separation of C9orf72 Dipeptide Repeats Perturbs Stress Granule Dynamics.

Liquid-liquid phase separation (LLPS) of RNA-binding proteins plays an important role in the formation of multiple membrane-less organelles involved in RNA metabolism, including stress granules. Defects in stress granule homeostasis constitute a cornerstone of ALS/FTLD pathogenesis. Polar residues (tyrosine and glutamine) have been previously demonstrated to be critical for phase separation of ALS-linked stress granule proteins. We now identify an active role for arginine-rich domains in these phase separations. Moreover, arginine-rich dipeptide repeats (DPRs) derived from C9orf72 hexanucleotide repeat expansions similarly undergo LLPS and induce phase separation of a large set of proteins involved in RNA and stress granule metabolism. Expression of arginine-rich DPRs in cells induced spontaneous stress granule assembly that required both eIF2α phosphorylation and G3BP. Together with recent reports showing that DPRs affect nucleocytoplasmic transport, our results point to an important role for arginine-rich DPRs in the pathogenesis of C9orf72 ALS/FTLD.

Stress-specific differences in assembly and composition of stress granules and related foci.

Cells have developed different mechanisms to respond to stress, including the formation of cytoplasmic foci known as stress granules (SGs). SGs are dynamic and formed as a result of stress-induced inhibition of translation. Despite enormous interest in SGs due to their contribution to the pathogenesis of several human diseases, many aspects of SG formation are poorly understood. SGs induced by different stresses are generally assumed to be uniform, although some studies suggest that different SG subtypes and SG-like cytoplasmic foci exist. Here, we investigated the molecular mechanisms of SG assembly and characterized their composition when induced by various stresses. Our data revealed stress-specific differences in composition, assembly and dynamics of SGs and SG-like cytoplasmic foci. Using a set of genetically modified haploid human cells, we determined the molecular circuitry of stress-specific translation inhibition upstream of SG formation and its relation to cell survival. Finally, our studies characterize cytoplasmic stress-induced foci related to, but distinct from, canonical SGs, and also introduce haploid cells as a valuable resource to study RNA granules and translation control mechanisms.

Mammalian ataxin-2 modulates translation control at the pre-initiation complex via PI3K/mTOR and is induced by starvation.

Ataxin-2 is a cytoplasmic protein, product of the ATXN2 gene, whose deficiency leads to obesity, while its gain-of-function leads to neural atrophy. Ataxin-2 affects RNA homeostasis, but its effects are unclear. Here, immunofluorescence analysis suggested that ataxin-2 associates with 48S pre-initiation components at stress granules in neurons and mouse embryonic fibroblasts, but is not essential for stress granule formation. Coimmunoprecipitation analysis showed associations of ataxin-2 with initiation factors, which were concentrated at monosome fractions of polysome gradients like ataxin-2, unlike its known interactor PABP. Mouse embryonic fibroblasts lacking ataxin-2 showed increased phosphorylation of translation modulators 4E-BP1 and ribosomal protein S6 through the PI3K-mTOR pathways. Indeed, human neuroblastoma cells after trophic deprivation showed a strong induction of ATXN2 transcript via mTOR inhibition. Our results support the notion that ataxin-2 is a nutritional stress-inducible modulator of mRNA translation at the pre-initiation complex.

Ebola Virus Does Not Induce Stress Granule Formation during Infection and Sequesters Stress Granule Proteins within Viral Inclusions.

UNLABELLED: A hallmark of Ebola virus (EBOV) infection is the formation of viral inclusions in the cytoplasm of infected cells. These viral inclusions contain the EBOV nucleocapsids and are sites of viral replication and nucleocapsid maturation. Although there is growing evidence that viral inclusions create a protected environment that fosters EBOV replication, little is known about their role in the host response to infection. The cellular stress response is an effective antiviral strategy that leads to stress granule (SG) formation and translational arrest mediated by the phosphorylation of a translation initiation factor, the α subunit of eukaryotic initiation factor 2 (eIF2α). Here, we show that selected SG proteins are sequestered within EBOV inclusions, where they form distinct granules that colocalize with viral RNA. These inclusion-bound (IB) granules are functionally and structurally different from canonical SGs. Formation of IB granules does not indicate translational arrest in the infected cells. We further show that EBOV does not induce formation of canonical SGs or eIF2α phosphorylation at any time postinfection but is unable to fully inhibit SG formation induced by different exogenous stressors, including sodium arsenite, heat, and hippuristanol. Despite the sequestration of SG marker proteins into IB granules, canonical SGs are unable to form within inclusions, which we propose might be mediated by a novel function of VP35, which disrupts SG formation. This function is independent of VP35's RNA binding activity. Further studies aim to reveal the mechanism for SG protein sequestration and precise function within inclusions. IMPORTANCE: Although progress has been made developing antiviral therapeutics and vaccines against the highly pathogenic Ebola virus (EBOV), the cellular mechanisms involved in EBOV infection are still largely unknown. To better understand these intracellular events, we investigated the cellular stress response, an antiviral pathway manipulated by many viruses. We show that EBOV does not induce formation of stress granules (SGs) in infected cells and is therefore unrestricted by their concomitant translational arrest. We identified SG proteins sequestered within viral inclusions, which did not impair protein translation. We further show that EBOV is unable to block SG formation triggered by exogenous stress early in infection. These findings provide insight into potential targets of therapeutic intervention. Additionally, we identified a novel function of the interferon antagonist VP35, which is able to disrupt SG formation.

YB-1 regulates tiRNA-induced Stress Granule formation but not translational repression.

Stress-induced angiogenin (ANG)-mediated tRNA cleavage promotes a cascade of cellular events that starts with production of tRNA-derived stress-induced RNAs (tiRNAs) and culminates with enhanced cell survival. This stress response program relies on a subset tiRNAs that inhibit translation initiation and induce the assembly of stress granules (SGs), cytoplasmic ribonucleoprotein complexes with cytoprotective and pro-survival properties. SG-promoting tiRNAs bear oligoguanine motifs at their 5'-ends, assemble G-quadruplex-like structures and interact with the translational silencer YB-1. We used CRISPR/Cas9-based genetic manipulations and biochemical approaches to examine the role of YB-1 in tiRNA-mediated translational repression and SG assembly. We found that YB-1 directly binds to tiRNAs via its cold shock domain. This interaction is required for packaging of tiRNA-repressed mRNAs into SGs but is dispensable for tiRNA-mediated translational repression. Our studies reveal the functional role of YB-1 in the ANG-mediated stress response program.

Vinca alkaloid drugs promote stress-induced translational repression and stress granule formation.

Resistance to chemotherapy drugs is a serious therapeutic problem and its underlying molecular mechanisms are complex. Stress granules (SGs), cytoplasmic ribonucleoprotein complexes assembled in cells exposed to stress, are implicated in various aspects of cancer cell metabolism and survival. SGs promote the survival of stressed cells by reprogramming gene expression and inhibiting pro-apoptotic signaling cascades. We show that the vinca alkaloid (VA) class of anti-neoplastic agents potently activates a SG-mediated stress response program. VAs inhibit translation initiation by simultaneous activation of eIF4E-BP1 and phosphorylation of eIF2α, causing polysome disassembly and SG assembly. VA-induced SGs contain canonical SG components but lack specific signaling molecules. Blocking VA-induced SG assembly by inactivating eIF4EBP1 or inhibiting eIF2α phosphorylation decreases cancer cell viability and promotes apoptosis. Our data describe previously unappreciated effects of VAs on cellular RNA metabolism and illuminate the roles of SGs in cancer cell survival.

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.