QKI shuttles internal m7G-modified transcripts into stress granules and modulates mRNA metabolism.

N7-methylguanosine (m7G) modification, routinely occurring at mRNA 5' cap or within tRNAs/rRNAs, also exists internally in messenger RNAs (mRNAs). Although m7G-cap is essential for pre-mRNA processing and protein synthesis, the exact role of mRNA internal m7G modification remains elusive. Here, we report that mRNA internal m7G is selectively recognized by Quaking proteins (QKIs). By transcriptome-wide profiling/mapping of internal m7G methylome and QKI-binding sites, we identified more than 1,000 high-confidence m7G-modified and QKI-bound mRNA targets with a conserved "GANGAN (N = A/C/U/G)" motif. Strikingly, QKI7 interacts (via C terminus) with the stress granule (SG) core protein G3BP1 and shuttles internal m7G-modified transcripts into SGs to regulate mRNA stability and translation under stress conditions. Specifically, QKI7 attenuates the translation efficiency of essential genes in Hippo signaling pathways to sensitize cancer cells to chemotherapy. Collectively, we characterized QKIs as mRNA internal m7G-binding proteins that modulate target mRNA metabolism and cellular drug resistance.

G3BPs tether the TSC complex to lysosomes and suppress mTORC1 signaling.

Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.

G3BP1 Is a Tunable Switch that Triggers Phase Separation to Assemble Stress Granules.

The mechanisms underlying ribonucleoprotein (RNP) granule assembly, including the basis for establishing and maintaining RNP granules with distinct composition, are unknown. One prominent type of RNP granule is the stress granule (SG), a dynamic and reversible cytoplasmic assembly formed in eukaryotic cells in response to stress. Here, we show that SGs assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein-RNA interaction network. The central node of this network is G3BP1, which functions as a molecular switch that triggers RNA-dependent LLPS in response to a rise in intracellular free RNA concentrations. Moreover, we show that interplay between three distinct intrinsically disordered regions (IDRs) in G3BP1 regulates its intrinsic propensity for LLPS, and this is fine-tuned by phosphorylation within the IDRs. Further regulation of SG assembly arises through positive or negative cooperativity by extrinsic G3BP1-binding factors that strengthen or weaken, respectively, the core SG network.

Structure-Mediated RNA Decay by UPF1 and G3BP1.

Post-transcriptional mechanisms regulate the stability and, hence, expression of coding and noncoding RNAs. Sequence-specific features within the 3' untranslated region (3' UTR) often direct mRNAs for decay. Here, we characterize a genome-wide RNA decay pathway that reduces the half-lives of mRNAs based on overall 3' UTR structure formed by base pairing. The decay pathway is independent of specific single-stranded sequences, as regulation is maintained in both the original and reverse complement orientation. Regulation can be compromised by reducing the overall structure by fusing the 3' UTR with an unstructured sequence. Mutating base-paired RNA regions can also compromise this structure-mediated regulation, which can be restored by re-introducing base-paired structures of different sequences. The decay pathway requires the RNA-binding protein UPF1 and its associated protein G3BP1. Depletion of either protein increased steady-state levels of mRNAs with highly structured 3' UTRs as well as highly structured circular RNAs. This structure-dependent mechanism therefore enables cells to selectively regulate coding and noncoding RNAs.

The force-dependent filamin A-G3BP1 interaction regulates phase-separated stress granule formation.

Filamin A (FLNA) is an actin crosslinking protein that mediates mechanotransduction. External and internal mechanical forces, through the actin cytoskeleton, can induce conformational changes of the FLNA molecule to expose cryptic binding sites for its binding partners. Here, we identified Ras GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) as a new FLNA mechanobinding partner. Unlike other FLNA binding partners to the mechanosensing domain repeat 21 (R21), G3BP1 requires an additional neighboring repeat R22 to interact. We demonstrated that their interaction occurs in the cytosol of living cells in an actin polymerization-dependent manner. We also mapped the FLNA-binding site on G3BP1 and found that a F360A point mutation in the RNA recognition motif disrupts the interaction. RNA interfered with the FLNA-G3BP1 interaction, and FLNA did not localize in RNA-rich stress granules (SGs). Disruption of the interaction was sufficient to promote phase-separated SG formation, and arsenite treatment further stimulated the formation of SGs. Taken together, these data identify G3BP1 as a new mechanobinding protein that interacts with the FLNA mechanosensing domain R21 and suggest that SG formation is partially regulated by mechanical force.

SIRT2 negatively regulates the cGAS-STING pathway by deacetylating G3BP1.

SIRT2, a cytoplasmic member of the Sirtuin family, has important roles in immunity and inflammation. However, its function in regulating the response to DNA virus infection remains elusive. Here, we find that SIRT2 is a unique regulator among the Sirtuin family that negatively modulates the cGAS-STING-signaling pathway. SIRT2 is down-regulated after Herpes simplex virus-1 (HSV-1) infection, and SIRT2 deficiency markedly elevates the expression levels of type I interferon (IFN). SIRT2 inhibits the DNA binding ability and droplet formation of cGAS by interacting with and deacetylating G3BP1 at K257, K276, and K376, leading to the disassembly of the cGAS-G3BP1 complex, which is critical for cGAS activation. Administration of AGK2, a selective SIRT2 inhibitor, protects mice from HSV-1 infection and increases the expression of IFN and IFN-stimulated genes. Our study shows that SIRT2 negatively regulates cGAS activation through G3BP1 deacetylation, suggesting a potential antiviral strategy by modulating SIRT2 activity.

Yin and yang regulation of stress granules by Caprin-1.

Stress granules (SGs) are cytoplasmic biomolecular condensates containing proteins and RNAs in response to stress. Ras-GTPase-activating protein binding protein 1 (G3BP1) is a core SG protein. Caprin-1 and ubiquitin specific peptidase 10 (USP10) interact with G3BP1, facilitating and suppressing SG formation, respectively. The crystal structures of the nuclear transport factor 2-like (NTF2L) domain of G3BP1 in complex with the G3BP1-interacting motif (GIM) of Caprin-1 and USP10 show that both GIMs bind to the same hydrophobic pocket of G3BP1. Moreover, both GIMs suppressed the liquid-liquid phase separation (LLPS) of G3BP1, suggesting that Caprin-1 likely facilitates SG formation via other mechanisms. Thus, we dissected various domains of Caprin-1 and investigated their role in LLPS in vitro and SG formation in cells. The C-terminal domain of Caprin-1 underwent spontaneous LLPS, whereas the N-terminal domain and GIM of Caprin-1 suppressed LLPS of G3BP1. The opposing effect of the N- and C-terminal domains of Caprin-1 on SG formation were demonstrated in cells with or without the endogenous Caprin-1. We propose that the N- and C-terminal domains of Caprin-1 regulate SG formation in a "yin and yang" fashion, mediating the dynamic and reversible assembly of SGs.

c-Jun N-Terminal Kinase Promotes Stress Granule Assembly and Neurodegeneration in C9orf72-Mediated ALS and FTD.

Stress granules are the RNA/protein condensates assembled in the cells under stress. They play a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, how stress granule assembly is regulated and related to ALS/FTD pathomechanism is incompletely understood. Mutation in the C9orf72 gene is the most common cause of familial ALS and FTD. C9orf72 mutation causes the formation of toxic dipeptide repeats. Here we show that the two most toxic dipeptide repeats [i.e., poly(GR) and poly(PR)] activate c-Jun N-terminal kinase (JNK) via the ER-stress response protein IRE1 using fly and cellular models. Further, we show that activated JNK promotes stress granule assembly in cells by promoting the transcription of one of the key stress granule proteins (i.e., G3BP1) by inducing histone 3 phosphorylation. Consistent with these findings, JNK or IRE1 inhibition reduced stress granule formation, histone 3 phosphorylation, G3BP1 mRNA and protein levels, and neurotoxicity in cells overexpressing poly(GR) and poly(PR) or neurons derived from male and female C9ALS/FTD patient-induced pluripotent stem cells. Our findings connect ER stress, JNK activation, and stress granule assembly in a unified pathway contributing to C9ALS/FTD neurodegeneration.SIGNIFICANCE STATEMENT c-Jun N-terminal kinase (JNK) is a part of the mitogen-activated protein kinase pathway, which is the central node for the integration of multiple stress signals. Cells are under constant stress in neurodegenerative diseases, and how these cells respond to stress signals is a critical factor in determining their survival or death. Previous studies have shown JNK as a major contributor to cellular apoptosis. Here, we show the role of JNK in stress granule assembly. We identify that toxic dipeptide repeats produced in ALS/FTD conditions activate JNK. The activated JNK in the nucleus can induce histone modifications which increase G3BP1 expression, thus promoting stress granule assembly and neurodegeneration.

African swine fever virus pS273R antagonizes stress granule formation by cleaving the nucleating protein G3BP1 to facilitate viral replication.

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs are regulated by different stimulators including viral infection, which is involved in the antiviral activity of host cells to limit viral propagation. To survive, several viruses have been reported to execute various strategies, such as modulating SG formation, to create optimal surroundings for viral replication. African swine fever virus (ASFV) is one of the most notorious pathogens in the global pig industry. However, the interplay between ASFV infection and SG formation remains largely unknown. In this study, we found that ASFV infection inhibited SG formation. Through SG inhibitory screening, we found that several ASFV-encoded proteins are involved in inhibition of SG formation. Among them, an ASFV S273R protein (pS273R), the only cysteine protease encoded by the ASFV genome, significantly affected SG formation. ASFV pS273R interacted with G3BP1 (Ras-GTPase-activating protein [SH3 domain] binding protein 1), a vital nucleating protein of SG formation. Furthermore, we found that ASFV pS273R cleaved G3BP1 at the G140-F141 to produce two fragments (G3BP1-N1-140 and G3BP1-C141-456). Interestingly, both the pS273R-cleaved fragments of G3BP1 lost the ability to induce SG formation and antiviral activity. Taken together, our finding reveals that the proteolytic cleavage of G3BP1 by ASFV pS273R is a novel mechanism by which ASFV counteracts host stress and innate antiviral responses.

A new tandem repeat-enriched lncRNA XLOC_008672 promotes gastric carcinogenesis by regulating G3BP1 expression.

Aberrant expression of forkhead box transcription factor 1 (FOXM1) plays critical roles in a variety of human malignancies and predicts poor prognosis. However, little is known about the crosstalk between FOXM1 and long noncoding RNAs (lncRNAs) in tumorigenesis. The present study identifies a previously uncharacterized lncRNA XLOC_008672 in gastric cancer (GC), which is regulated by FOXM1 and possesses multiple copies of tandem repetitive sequences. LncRNA microarrays are used to screen differentially expressed lncRNAs in FOXM1 knockdown GC cells, and then the highest fold downregulation lncRNA XLOC_008672 is screened out. Sequence analysis reveals that the new lncRNA contains 62 copies of 37-bp tandem repeats. It is transcriptionally activated by FOXM1 and functions as a downstream effector of FOXM1 in GC cells through in vitro and in vivo functional assays. Elevated expression of XLOC_008672 is found in GC tissues and indicates worse prognosis. Mechanistically, XLOC_008672 can bind to small nuclear ribonucleoprotein polypeptide A (SNRPA), thereby enhancing mRNA stability of Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) and, consequently, facilitating GC cell proliferation and migration. Our study discovers a new uncharacterized lncRNA XLOC_008672 involved in GC carcinogenesis and progression. Targeting FOXM1/XLOC_008672/SNRPA/G3BP1 signaling axis might be a promising therapeutic strategy for GC.

Translational arrest and mRNA decay are independent activities of alphaherpesvirus virion host shutoff proteins.

The herpes simplex virus 1 (HSV1) virion host shutoff (vhs) protein is an endoribonuclease that regulates the translational environment of the infected cell, by inducing the degradation of host mRNA via cellular exonuclease activity. To further understand the relationship between translational shutoff and mRNA decay, we have used ectopic expression to compare HSV1 vhs (vhsH) to its homologues from four other alphaherpesviruses - varicella zoster virus (vhsV), bovine herpesvirus 1 (vhsB), equine herpesvirus 1 (vhsE) and Marek's disease virus (vhsM). Only vhsH, vhsB and vhsE induced degradation of a reporter luciferase mRNA, with poly(A)+ in situ hybridization indicating a global depletion of cytoplasmic poly(A)+ RNA and a concomitant increase in nuclear poly(A)+ RNA and the polyA tail binding protein PABPC1 in cells expressing these variants. By contrast, vhsV and vhsM failed to induce reporter mRNA decay and poly(A)+ depletion, but rather, induced cytoplasmic G3BP1 and poly(A)+ mRNA- containing granules and phosphorylation of the stress response proteins eIF2α and protein kinase R. Intriguingly, regardless of their apparent endoribonuclease activity, all vhs homologues induced an equivalent general blockade to translation as measured by single-cell puromycin incorporation. Taken together, these data suggest that the activities of translational arrest and mRNA decay induced by vhs are separable and we propose that they represent sequential steps of the vhs host interaction pathway.

Novel stress granule-like structures are induced via a paracrine mechanism during viral infection.

To rapidly adapt to stresses such as infections, cells have evolved several mechanisms, which include the activation of stress response pathways and the innate immune response. These stress responses result in the rapid inhibition of translation and condensation of stalled mRNAs with RNA-binding proteins and signalling components into cytoplasmic biocondensates called stress granules (SGs). Increasing evidence suggests that SGs contribute to antiviral defence, and thus viruses need to evade these responses to propagate. We previously showed that feline calicivirus (FCV) impairs SG assembly by cleaving the scaffolding protein G3BP1. We also observed that uninfected bystander cells assembled G3BP1-positive granules, suggesting a paracrine response triggered by infection. We now present evidence that virus-free supernatant generated from infected cells can induce the formation of SG-like foci, which we name paracrine granules. They are linked to antiviral activity and exhibit specific kinetics of assembly-disassembly, and protein and RNA composition that are different from canonical SGs. We propose that this paracrine induction reflects a novel cellular defence mechanism to limit viral propagation and promote stress responses in bystander cells.

Targeting the NTF2-like domain of G3BP1: Novel modulators of intracellular granule dynamics.

Stress granule (SG) is a temporary cellular structure that plays a crucial role in the regulation of mRNA and protein sequestration during various cellular stress conditions. SG enables cells to cope with stress more effectively, conserving vital energy and resources. Focusing on the NTF2-like domain of G3BP1, a key protein in SG dynamics, we explore to identify and characterize novel small molecules involved in SG modulation without external stressors. Through in silico molecular docking approach to simulate the interaction between various compounds and the NTF2-like domain of G3BP1, we identified three compounds as potential candidates that could bind to the NTF2-like domain of G3BP1. Subsequent immunofluorescence experiments demonstrated that these compounds induce the formation of SG-like, G3BP1-positive granules. Importantly, the granule formation by these compounds occurs independent from the phosphorylation of eIF2α, a common mechanism in SG formation, suggesting that it might offer a new strategy for influencing SG dynamics implicated in various diseases.

A closer look at mammalian antiviral condensates.

Several biomolecular condensates assemble in mammalian cells in response to viral infection. The most studied of these are stress granules (SGs), which have been proposed to promote antiviral innate immune signaling pathways, including the RLR-MAVS, the protein kinase R (PKR), and the OAS-RNase L pathways. However, recent studies have demonstrated that SGs either negatively regulate or do not impact antiviral signaling. Instead, the SG-nucleating protein, G3BP1, may function to perturb viral RNA biology by condensing viral RNA into viral-aggregated RNA condensates, thus explaining why viruses often antagonize G3BP1 or hijack its RNA condensing function. However, a recently identified condensate, termed double-stranded RNA-induced foci, promotes the activation of the PKR and OAS-RNase L antiviral pathways. In addition, SG-like condensates known as an RNase L-induced bodies (RLBs) have been observed during many viral infections, including SARS-CoV-2 and several flaviviruses. RLBs may function in promoting decay of cellular and viral RNA, as well as promoting ribosome-associated signaling pathways. Herein, we review these recent advances in the field of antiviral biomolecular condensates, and we provide perspective on the role of canonical SGs and G3BP1 during the antiviral response.

The stress granule protein G3BP1 promotes pre-condensation of cGAS to allow rapid responses to DNA.

Cyclic GMP-AMP synthase (cGAS) functions as a key sensor for microbial invasion and cellular damage by detecting emerging cytosolic DNA. Here, we report that GTPase-activating protein-(SH3 domain)-binding protein 1 (G3BP1) primes cGAS for its prompt activation by engaging cGAS in a primary liquid-phase condensation state. Using high-resolution microscopy, we show that in resting cells, cGAS exhibits particle-like morphological characteristics, which are markedly weakened when G3BP1 is deleted. Upon DNA challenge, the pre-condensed cGAS undergoes liquid-liquid phase separation (LLPS) more efficiently. Importantly, G3BP1 deficiency or its inhibition dramatically diminishes DNA-induced LLPS and the subsequent activation of cGAS. Interestingly, RNA, previously reported to form condensates with cGAS, does not activate cGAS. Accordingly, we find that DNA - but not RNA - treatment leads to the dissociation of G3BP1 from cGAS. Taken together, our study shows that the primary condensation state of cGAS is critical for its rapid response to DNA.

The stress granule protein G3BP1 alleviates spinocerebellar ataxia-associated deficits.

Polyglutamine diseases are a group of neurodegenerative disorders caused by an abnormal expansion of CAG repeat tracts in the codifying regions of nine, otherwise unrelated, genes. While the protein products of these genes are suggested to play diverse cellular roles, the pathogenic mutant proteins bearing an expanded polyglutamine sequence share a tendency to self-assemble, aggregate and engage in abnormal molecular interactions. Understanding the shared paths that link polyglutamine protein expansion to the nervous system dysfunction and the degeneration that takes place in these disorders is instrumental to the identification of targets for therapeutic intervention. Among polyglutamine diseases, spinocerebellar ataxias (SCAs) share many common aspects, including the fact that they involve dysfunction of the cerebellum, resulting in ataxia. Our work aimed at exploring a putative new therapeutic target for the two forms of SCA with higher worldwide prevalence, SCA type 2 (SCA2) and type 3 (SCA3), which are caused by expanded forms of ataxin-2 (ATXN2) and ataxin-3 (ATXN3), respectively. The pathophysiology of polyglutamine diseases has been described to involve an inability to properly respond to cell stress. We evaluated the ability of GTPase-activating protein-binding protein 1 (G3BP1), an RNA-binding protein involved in RNA metabolism regulation and stress responses, to counteract SCA2 and SCA3 pathology, using both in vitro and in vivo disease models. Our results indicate that G3BP1 overexpression in cell models leads to a reduction of ATXN2 and ATXN3 aggregation, associated with a decrease in protein expression. This protective effect of G3BP1 against polyglutamine protein aggregation was reinforced by the fact that silencing G3bp1 in the mouse brain increases human expanded ATXN2 and ATXN3 aggregation. Moreover, a decrease of G3BP1 levels was detected in cells derived from patients with SCA2 and SCA3, suggesting that G3BP1 function is compromised in the context of these diseases. In lentiviral mouse models of SCA2 and SCA3, G3BP1 overexpression not only decreased protein aggregation but also contributed to the preservation of neuronal cells. Finally, in an SCA3 transgenic mouse model with a severe ataxic phenotype, G3BP1 lentiviral delivery to the cerebellum led to amelioration of several motor behavioural deficits. Overall, our results indicate that a decrease in G3BP1 levels may be a contributing factor to SCA2 and SCA3 pathophysiology, and that administration of this protein through viral vector-mediated delivery may constitute a putative approach to therapy for these diseases, and possibly other polyglutamine disorders.

A novel G3BP1-GFP reporter human lung cell system enabling real-time monitoring of stress granule dynamics for in vitro lung toxicity assessment.

Under various cellular stress conditions, including exposure to toxic chemicals, RNA-binding proteins (RBPs), including Ras GTPase-activating protein-binding protein 1 (G3BP1), aggregate and form stress granule complexes, which serve as hallmarks of cellular stress. The existing methods for analyzing stress granule assembly have limitations in the rapid detection of dynamic cellular stress and ignore the effects of constitutively overexpressed RBP on cellular stress and stress-related processes. Therefore, to overcome these limitations, we established a G3BP1-GFP reporter in a human lung epithelial cell line using CRISPR/Cas9-based knock-in as an alternative system for stress granule analysis. We showed that the G3BP1-GFP reporter system responds to stress conditions and forms a stress granule complex similar to that of native G3BP1. Furthermore, we validated the stress granule response of an established cell line under exposure to various household chemicals. Overall, this novel G3BP1-GFP reporter human lung cell system is capable of monitoring stress granule dynamics in real time and can be used for assessing the lung toxicity of various substances in vitro.

Stress Granule Core Protein-Derived Peptides Inhibit Assembly of Stress Granules and Improve Sorafenib Sensitivity in Cancer Cells.

Upon a variety of environmental stresses, eukaryotic cells usually recruit translational stalled mRNAs and RNA-binding proteins to form cytoplasmic condensates known as stress granules (SGs), which minimize stress-induced damage and promote stress adaptation and cell survival. SGs are hijacked by cancer cells to promote cell survival and are consequently involved in the development of anticancer drug resistance. However, the design and application of chemical compounds targeting SGs to improve anticancer drug efficacy have rarely been studied. Here, we developed two types of SG inhibitory peptides (SIPs) derived from SG core proteins Caprin1 and USP10 and fused with cell-penetrating peptides to generate TAT-SIP-C1/2 and SIP-U1-Antp, respectively. We obtained 11 SG-inducing anticancer compounds from cell-based screens and explored the potential application of SIPs in overcoming resistance to the SG-inducing anticancer drug sorafenib. We found that SIPs increased the sensitivity of HeLa cells to sorafenib via the disruption of SGs. Therefore, anticancer drugs which are competent to induce SGs could be combined with SIPs to sensitize cancer cells, which might provide a novel therapeutic strategy to alleviate anticancer drug resistance.

Effect of P53 nuclear localization mediated by G3BP1 on ferroptosis in acute liver failure.

This study investigated whether G3BP1 could regulate ferroptosis in hepatocytes during ALF by affecting the entry of P53 into the nucleus. Promoting G3BP1 expression could inhibit P53 entry by binding to the nuclear localization sequence of P53. The inhibition of SLC7A11 transcription was weakened after blocking of P53 binding to the promoter region of the SLC7A11 gene. The SLC7A11-GSH-GPX4 antiferroptotic pathway was subsequently activated, and the level of ferroptosis in ALF hepatocytes was inhibited.

RNA partitioning into stress granules is based on the summation of multiple interactions.

Stress granules (SGs) are stress-induced RNA-protein assemblies formed from a complex transcriptome of untranslating ribonucleoproteins (RNPs). Although RNAs can be either enriched or depleted from SGs, the rules that dictate RNA partitioning into SGs are unknown. We demonstrate that the SG-enriched NORAD RNA is sufficient to enrich a reporter RNA within SGs through the combined effects of multiple elements. Moreover, artificial tethering of G3BP1, TIA1, or FMRP can target mRNAs into SGs in a dose-dependent manner with numerous interactions required for efficient SG partitioning, which suggests individual protein interactions have small effects on the SG partitioning of mRNPs. This is supported by the observation that the SG transcriptome is largely unchanged in cell lines lacking the abundant SG RNA-binding proteins G3BP1 and G3BP2. We suggest the targeting of RNPs into SGs is due to a summation of potential RNA-protein, protein-protein, and RNA-RNA interactions with no single interaction dominating RNP recruitment into SGs.