G3BP1 is a multi-functional protein that is best known for its role in the assembly and dynamics of stress granules. Recent studies have highlighted that G3BP1 also has other functions related to RNA metabolism. In the context of disease, G3BP1 has been therapeutically targeted in cancers because its overexpression is correlated with proliferation of cancerous cells and metastasis. However, evidence suggests that G3BP1 is essential for neuronal development and possibly neuronal maintenance. In this review, we will examine the many functions that are carried out by G3BP1 in the context of neurons and speculate how these functions are critical to the progression of neurodegenerative diseases. Additionally, we will highlight the similarities and differences between G3BP1 and the closely related protein G3BP2, which is frequently overlooked. Although G3BP1 and G3BP2 have both been deemed important for stress granule assembly, their roles may differ in other cellular pathways, some of which are specific to the CNS, and presents an opportunity for further exploration.

Keywords: ALS; G3BP1; RNA binding proteins; mRNA stability; neurodegenerative disease; translation.

Background: The objective of this study was to investigate the effects of glycaemic variability (GV) on intimal hyperplasia and plaque stability after coronary stenting via autophagy-mediated G3BP1/NLRP3 inflammasome signalling.

Methods: In the clinical study, between July 2017 and December 2017, 95 patients with acute myocardial infarction (AMI) and diabetes mellitus (DM) comorbidity received stent implantation. The patients were followed up for 2 years after discharge. The patients were divided into a low-GV (n=61) and high-GV (n=34) group, and the incidence of recurrent AMI was measured. In the animal study, thirteen pigs were divided into a sham (n=3), low-GV DM (n=5) and high-GV DM group (n=5). Intima samples were analysed by optical coherence tomography 22 weeks after coronary stenting. Becn1, LC3B, p62, G3BP1 and NLRP3 protein levels in the intima were examined by western blot. In vitro experiments with THP-1 cells were also conducted.

Results: In the high-GV group, patients exhibited a higher recurrent AMI, greater neointimal thickness, increased p62 and NLRP3 expression, and decreased Becn1, LC3B and G3BP1 expression compared with the low-GV group (P<0.05). The effects of high GV could be abolished by rapamycin but were aggravated by 3-methyladenine.

Conclusions: GV might impact the intimal hyperplasia and plaque stability via autophagy-mediated G3BP1/NLRP3 inflammasome signalling. GV and the autophagy-mediated G3BP1/NLRP3 inflammasome may be promising targets for the treatment of coronary heart disease.

Keywords: G3BP1; Glycaemic variability (GV); NLRP3 inflammasome; autophagy; coronary heart disease (CHD); diabetes; intima; plaque stability.

In myeloid neoplasms, deletions of the long arm of chromosome 5 del(5q) and 7 (-7/del(7q) ) are common karyotypic abnormalities. The concurrence of del(5q) and -7/del(7q) accounts for poor prognosis in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Comprehensive analysis of copy number abnormalities and genetic mutations related to del(5q) and -7/del(7q) revealed previously cryptic pathophysiology, leading to frequent hemizygous/homozygous mutations and haploinsufficiency. In addition, detailed somatic mutations on chr5q were detected using whole-exome sequencing. CSNK1A1 and G3BP1 are located within the common deleted regions (CDRs) (5q31.1-5q33.1), and another driver gene DDX41 is present in the more telomeric region (5q35.3). All the genes mentioned above exhibited haploinsufficiency because of deletions, and low expression of G3BP1 and DDX41 correlated with poor survival. The related mutational events outside of chr5q, TP53 mutation is most frequently observed in del(5q) cases. Regarding -7/del(7q), 3 CDRs were located in 7q22, 7q34, and 7q35-36. Somatic mutations of the corresponding genes to each CDR (CUX1: 7q22, LUC7L2: 7q34, EZH2: 7q35-36) were identified, indicating that the loss of function or haploinsufficiency might result in the downstream pathological consequences. These recent findings have remarkably offered insights into genetic and clinical consequences in MDS/AML cases with del(5q) and -7/del(7q).

Background: Pancreatic cancer is a malignant tumor and its incidence has increased in recent years. Carboxypeptidase E (CPE) is a prohormone/proneuropeptide processing enzyme that has been shown to be associated with tumor growth and invasion in various cancers including pancreatic cancer.

Objective: To understand the molecular mechanism underlying the proliferative effects of CPE in cancer cells.

Methods: We down-regulated CPE gene expression in PANC-1 cell, a pancreatic cell line, and investigated mRNA, miRNA, circRNA and lncRNA expression profiling in PANC-1 cells from control group and CPE knock-down group by microarray analysis. We further validated the top 14 differentially expressed circRNAs by qRT-PCR.

Results: Our results showed that CPE down-regulation caused decreased cell proliferation. The microarray data showed 107, 15, 299 and 360 differentially expressed mRNAs, miRNAs, circRNAs, and lncRNAs, respectively between control group and CPE knock-down group. Of Which, 41 mRNAs, 12 miRNAs, 133 circRNAs, and 262 lncRNAs were down-regulated; 66 mRNAs, 3 miRNAs, 166 circRNAs, and 98 lncRNAs were up-regulated. Bioinformatics analysis showed that the top significantly enriched pathways for the differentially expressed RNAs were related to cancer onset and/or progression, these included p53 signaling pathway, ECM-receptor interaction, focal adhesion and Wnt signaling pathway. We further performed network analysis to assess the mRNA, miRNA, circRNA and lncRNA correlations, and showed that HUWE1, hsa-miR-6780b-5p, has_circ_0058208 and lnc-G3BP1-3:8 were in the core position of the network.

Conclusions: Taken together, these results identified potential CPE regulated core genes and pathways for cell proliferation in pancreatic cancer cell, and therefore provide potential targets for the treatment of pancreatic cancer.

Keywords: Carboxypeptidase E; Pancreatic cancer; ceRNA analysis.

Background: Homer scaffolding protein 1 (Homer1) is a postsynaptic scaffold protein that regulates the structure and function of excitatory synaptic as well as its intracellular signal transduction. However, the role of Homer1 in colorectal cancer as well as the underlying molecular mechanisms has not been elucidated.

Materials and methods: To evaluate the alternations of gene expression during colorectal cancer, Homer1 expression was analyzed using the gene expression profiling interactive analysis and Oncomine analyses. The prognostic value of Homer1 expression was validated by our own colorectal cancer specimens using RT-PCR. Then, the cell viability, migration and invasion of colorectal cancer cell lines were detected by CCK-8 and transwell assay.

Results: We obtained the following important results. (1) Homer1 expression was significantly higher in colorectal cancer than normal samples. (2) Among patients with colorectal cancer, those with higher Homer1 expression had a lower survival rate. (3) The major mutation type of Homer1 in colorectal cancer samples was missense mutation. (4) Homer1 was able to promote colorectal cancer cell proliferation, migration, and invasion through up-regulating G3BP1 in vitro.

Conclusion: Our findings suggest that Homer1 may play a role in malignancy of colorectal cancer mainly through the G3BP1 signaling pathway, which might be a potential indicator of poor prognosis.

Keywords: G3BP1; Homer1; colorectal cancer; prognosis.

Glioblastoma (GBM) is the most common primary malignant brain tumor, and it has a uniformly poor prognosis. Hypoxia is a feature of the GBM microenvironment, and previous work has shown that cancer cells residing in hypoxic regions resist treatment. Hypoxia can trigger the formation of stress granules (SGs), sites of mRNA triage that promote cell survival. A screen of 1120 FDA-approved drugs identified 129 candidates that delayed the dissolution of hypoxia-induced SGs following a return to normoxia. Amongst these candidates, the selective estrogen receptor modulator (SERM) raloxifene delayed SG dissolution in a dose-dependent manner. SG dissolution typically occurs by 15 min post-hypoxia, however pre-treatment of immortalized U251 and U3024 primary GBM cells with raloxifene prevented SG dissolution for up to 2 h. During this raloxifene-induced delay in SG dissolution, translational silencing was sustained, eIF2α remained phosphorylated and mTOR remained inactive. Despite its well-described role as a SERM, raloxifene-mediated delay in SG dissolution was unaffected by co-administration of β-estradiol, nor did β-estradiol alone have any effect on SGs. Importantly, the combination of raloxifene and hypoxia resulted in increased numbers of late apoptotic/necrotic cells. Raloxifene and hypoxia also demonstrated a block in late autophagy similar to the known autophagy inhibitor chloroquine (CQ). Genetic disruption of the SG-nucleating proteins G3BP1 and G3BP2 revealed that G3BP1 is required to sustain the raloxifene-mediated delay in SG dissolution. Together, these findings indicate that modulating the stress response can be used to exploit the hypoxic niche of GBM tumors, causing cell death by disrupting pro-survival stress responses and control of protein synthesis.

Withaferin A (WA) is a steroidal lactone extracted from Withania somnifera, commonly known as Ashwagandha. WA has several therapeutic benefits. The current study aims to identify proteins that are potentially regulated by WA in prostate cancer (PCA) cells. We used a SILAC-based proteomic approach to analyze the expression of proteins in response to WA treatment at 4 h and 24 h time points in three PCA cell lines: 22Rv1, DU-145, and LNCaP. Ontology analysis suggested that prolonged treatment with WA upregulated the expression of proteins involved in stress-response pathways. Treatment with WA increased oxidative stress, reduced global mRNA translation, and elevated the expression of cytoprotective stress granule (SG) protein G3BP1. WA treatment also enhanced the formation of SGs. The elevated expression of G3BP1 and the formation of SGs might constitute a mechanism of cytoprotection in PCA cells. Knockdown of G3BP1 blocked SG formation and enhanced the efficacy of WA to reduce PCA cell survival. SIGNIFICANCE: Withaferin A, a steroidal lactone, extracted from Withania somniferum is a promising anti-cancer drug. Using a SILAC-based quantitative proteomic approach, we identified proteins changed by WA-treatment at 4 h and 24 h in three prostate cancer (PCA) cell lines. WA-treatment induced the expression of proteins involved in apoptosis and reduced the expression of proteins involved in cell growth at 4 h. WA-treatment for 24 h enhanced the expression of proteins involved in stress response pathways. WA-treated cells exhibited increased oxidative stress, reduced mRNA translation and enhanced SG formation. PCA is characterized by higher metabolic rate and increased oxidative stress. PCA with a higher stress tolerance can effectively adapt to anti-cancer treatment stress, leading to drug resistance and cellular protection. Enhancing the level of oxidative stress along with inhibition of corresponding cytoprotective stress response pathways is a feasible option to prevent PCA from getting adapted to treatment stress. WA-treatment induced oxidative stress, in combination with blocking SGs by G3BP1 targeting, offers a therapeutic strategy to reduce PCA cell survival.

Keywords: G3BP1; Natural compounds; Oxidative stress; Prostate cancer; Stress granule; Withaferin A.

Aims: The ubiquilin-like protein ubiquilin 2 (UBQLN2) is associated with amyotrophic lateral sclerosis and frontotemporal degeneration (ALS/FTD). The biological function of UBQLN2 has previously been shown to be related to stress granules (SGs). In this study, we aimed to clarify the regulatory relationship between UBQLN2 and SGs.

Methods: In this study, we transfected UBQLN2-WT or UBQLN2-P497H plasmids into cell lines (HEK293T, HeLa), and observed the process of SG dynamics by immunofluorescence. Meanwhile, immunoblot analyses the protein changes of stress granules related components.

Results: We observed that ubiquilin 2 colocalizes with the SG component proteins G3BP1, TIA-1, ATXN2, and PABPC1. In cells expressing WT UBQLN2 or P497H mutants, in the early stages of SG formation under oxidative stress, the percentage of cells with SGs and the number of SGs per cell decreased to varying degrees. Between WT and mutant, there was no significant difference in eIF2α activity after stress treatment. Interestingly, the UBQLN2 P497H mutant downregulates the level of TIA-1. In addition, the overexpression of the UBQLN2 P497H mutant inhibited the phosphorylation of 4E-BP1 and affected the nucleoplasmic distribution of TDP-43.

Conclusions: Ubiquilin 2 colocalizes with the SG component proteins G3BP1, TIA-1, ATXN2, and PABPC1. It participates in regulating SG dynamics. And UBQLN2 mutation affects the assembly of stress granules by regulating TIA-1. In addition, the overexpression of the UBQLN2 P497H mutant inhibited the phosphorylation of 4E-BP1 and affected the nuclear and cytoplasmic distribution of TDP-43. These provide new insights into the role of UBQLN2 in oxidative stress and the pathogenesis of ALS.

Keywords: TIA-1; UBQLN2; amyotrophic lateral sclerosis; protein aggregation; stress granules.

Small-cell lung cancer (SCLC) is a devastating subtype of lung cancer with few therapeutic options. Despite the advent of immunotherapy, platinum-based chemotherapy is still the irreplaceable first-line therapy for SCLCs. However, drug resistance will invariably occur in most patients and the outcomes are heterogeneous. Therefore, clinically feasible classification strategies and potential therapeutic targets for overcoming chemotherapy resistance are urgently needed. N⁶-methyladenosine (m⁶A) is a novel epigenetic decisive factor that is involved in tumor progression and drug resistance. However, almost nothing is known about m⁶A modification in SCLC. Here, we assessed 200 SCLC samples from patients who underwent chemotherapy from three different cohorts, including a validation cohort containing 71 cases with qPCR data and an independent cohort containing 79 cases with immunohistochemistry data (quantified as H-score). We systematically characterized the predictive landscape of m⁶A regulators in SCLC patients following with chemotherapy. Using the LASSO Cox model, we built a seven-regulator-based (ZCCHC4, IGF2BP3, ALKBH5, YTHDF3, METTL5, G3BP1, and RBMX) chemotherapy benefit predictive classifier (m⁶A score) and subsequently validated the classifier in two other cohorts. Time-dependent ROC and C-index analyses showed that the m⁶A score to possessed superior predictive power for chemotherapy benefit in comparison with other clinicopathological parameters. A multicohort multivariate analysis revealed that the m⁶A score is an independent factor that affects survival benefit across multiple cohorts. Our in vitro experimental results revealed that three regulators-ZCCHC4, G3BP1, and RBMX-may serve as promising novel therapeutic targets for overcoming chemoresistance in SCLCs. Our results, for the first time, demonstrate the predictive significance of m⁶A regulators for chemotherapy benefit, as well as their potential as therapeutic targets for overcoming chemotherapy resistance in SCLC patients. The m⁶A score was found to be a reliable prognostic tool that may help guide chemotherapy decisions for patients with SCLC.

Keywords: Chemotherapy resistance; Epigenetic modification; Individualized medicine; Small-cell lung cancer; m6A regulators.

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.

Keywords: G3BP1; LLPS; cGAS.

The nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2) is a critical viral protein that suppresses host gene expression by blocking the assembly of the ribosome on host messenger RNAs (mRNAs). To understand the mechanism of inhibition of host gene expression, we sought to identify cellular proteins that interact with nsp1. Using proximity-dependent biotinylation followed by proteomic analyses of biotinylated proteins, here we captured multiple dynamic interactions of nsp1 with host cell proteins. In addition to ribosomal proteins, we identified several pre-mRNA processing proteins that interact with nsp1, including splicing factors and transcription termination proteins, as well as exosome, and stress granule (SG)-associated proteins. We found that the interactions with transcription termination factors are primarily governed by the C-terminal region of nsp1 and are disrupted by the mutation of amino acids K164 and H165 that are essential for its host shutoff function. We further show that nsp1 interacts with Ras-GTPase-activating protein SH3-domain-binding protein 1 (G3BP1) and colocalizes with G3BP1 in SGs under sodium arsenite-induced stress. Finally, we observe that the presence of nsp1 disrupts the maturation of SGs over a long period. Isolation of SG core at different times shows a gradual loss of G3BP1 in the presence of nsp1.

Keywords: BioID2; SARS-CoV; SARS-CoV-2; nsp1; stress granule.

The tuberous sclerosis protein complex (TSC complex) is a key integrator of metabolic signals and cellular stress. In response to nutrient shortage and stresses, the TSC complex inhibits the mechanistic target of rapamycin complex 1 (mTORC1) at the lysosomes. mTORC1 is also inhibited by stress granules (SGs), RNA-protein assemblies that dissociate mTORC1. The mechanisms of lysosome and SG recruitment of mTORC1 are well studied. In contrast, molecular details on lysosomal recruitment of the TSC complex have emerged only recently. The TSC complex subunit 1 (TSC1) binds lysosomes via phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2]. The SG assembly factors 1 and 2 (G3BP1/2) have an unexpected lysosomal function in recruiting TSC2 when SGs are absent. In addition, high density lipoprotein binding protein (HDLBP, also named Vigilin) recruits TSC2 to SGs under stress. In this mini-review, we integrate the molecular mechanisms of lysosome and SG recruitment of the TSC complex. We discuss their interplay in the context of cell proliferation and migration in cancer and in the clinical manifestations of tuberous sclerosis complex disease (TSC) and lymphangioleiomyomatosis (LAM).

Keywords: G3BP1 (G3BP stress granule assembly factor 1); HDLBP; TSC complex; autophagy; lymphangioleiomyomatosis (LAM); lysosomes; mTORC1 (mechanistic target of rapamycin complex 1); stress granules (SG).

Cancer treatments are constantly evolving with new approaches to improve patient outcomes. Despite progresses, too many patients remain refractory to treatment due to either the development of resistance to therapeutic drugs and/or metastasis occurrence. Growing evidence suggests that these two barriers are due to transient survival mechanisms that are similar to those observed during stress response. We review the literature and current available open databases to study the potential role of stress response and, most particularly, the involvement of Stress Granules (proteins) in cancer. We propose that Stress Granule proteins may have prognostic value for patients.

Keywords: CAPRIN-1; G3BP1; G3BP2; TIA-1; TIAR; USP10; biomarker; cancer prognosis; cell death; metastasis; pro-survival properties; resistance; stress granules.

Translation arrest is a part of the cellular stress response that decreases energy consumption and enables rapid reprioritisation of gene expression. Often translation arrest leads to condensation of untranslated messenger ribonucleoproteins (mRNPs) into stress granules (SGs). Studies into mechanisms of SG formation and functions are complicated because various types of stress cause formation of SGs with different properties and composition. In this work we focused on the mechanism of SG formation triggered by UV damage. We demonstrate that UV-induced inhibition of translation does not involve inhibition of the mechanistic target of rapamycin (mTOR) signaling or dissociation of the 48S preinitiation complexes. The general control non-derepressible 2 (GCN2) kinase contributes to UV-induced SG formation, which is independent of the phosphorylation of the eukaryotic translation initiation factor 2α. Like many other types of SGs, condensation of UV-induced granules requires the Ras-GTPase-Activating Protein SH3-Domain-Binding Protein 1 (G3BP1). Our work reveals that in UV-treated cells the mechanisms of translation arrest and SG formation may be unlinked, resulting in SGs that do not contain the major type of polysome-free preinitiation complexes that accumulate in the cytoplasm.

Keywords: G3BP1; GCN2; MTOR; Stress granule; UVC.

Background: G3BP1 is a prognostic biomarker for many types of cancers; however, its role in oral squamous cell carcinoma remains unclear. We investigated the role of G3BP1 as a potential biomarker for proliferation, apoptosis and prognosis in oral squamous cell carcinoma.

Methods: We obtained samples of normal oral mucosa (n = 17), of oral squamous cell carcinoma tissues (n = 61) and paired adjacent tissues (n = 47) from Xiangya Hospital for immunohistochemical evaluation to measure the expression of G3BP1, E-cadherin, Ki67, and Cleaved-caspase3. Using data from The Cancer Genome Atlas, we performed bioinformatics analysis to investigate the prognosis, functions, signaling pathways, and immune infiltrate significance related to G3BP1 in oral squamous cell carcinoma.

Results: The G3BP1 protein level was significantly upregulated in oral squamous cell carcinoma tissues and was also positively associated with Ki67 and negatively associated with Cleaved-caspase3. Based on information available in online database, the G3BP1 mRNA level was significantly higher in oral squamous cell carcinoma than in normal tissues. High G3BP1 mRNA levels were associated with poor overall survival rates in patients with oral squamous cell carcinoma. Enrichment analysis showed that G3BP1 was involved in the helicase/catalytic/ATPase activity functions and spliceosome/RNA transport/ cell cycle pathways. Furthermore, G3BP1 mRNA levels were positively associated with CD4⁺ T cell infiltration.

Conclusions: G3BP1 may serve as a potential biomarker for proliferation, apoptosis, and prognosis of oral squamous cell carcinoma.

Keywords: Apoptosis; G3BP1; Oral squamous cell carcinoma; Prognosis; Proliferation.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing system has been broadly adopted for high-throughput genetic screens. However, the application of genome-wide single guide RNA (sgRNA) libraries can be challenging. We generated a custom sgRNA library, an order of magnitude smaller than genome-wide alternatives, to facilitate the genetic screening of RNA binding proteins (RBPs). We demonstrated the utility of our reagent in a genetic screen for RBPs that conveyed cellular resistance or sensitivity to oxidative stress induced by paraquat. This identified that CSDE1 and STRAP, proteins that interact with each other, convey sensitivity to oxidative stress and that Pumilio homologues (PUM1 and PUM2) convey resistance. Targeting eIF4-E1 and -A1 protected cells from high-dose paraquat, whereas eIF4E2 targeted cells did less well. We also found that G3BP1 promoted sensitivity to a low dose of paraquat but protected cells at a higher dose. Our study highlights the use of genetic screens to identify roles of RBPs and identifies novel genes regulating sensitivity to oxidative stress.

Nuclear Factor 90 (NF90) is a novel virus sensor that serves to initiate antiviral innate immunity by triggering the stress granules (SGs) formation. However, the regulation of the NF90-SGs pathway remain largely unclear. We found that Tim-3, an immune checkpoint inhibitor, promotes the ubiquitination and degradation of NF90 and inhibits NF90-SGs mediated antiviral immunity. Vesicular Stomatitis Virus (VSV) infection induces the up-regulation and activation of Tim-3 in macrophages which in turn recruited the E3 ubiquitin ligase TRIM47 to the zinc finger domain of NF90 and initiated a proteasome-dependent degradation via K48-linked ubiquitination at Lys297. Targeted inactivation of the Tim-3 enhances the NF90 downstream SGs formation by selectively increasing the phosphorylation of PKR and eIF2a, the expression of SGs markers G3BP1 and TIA-1, and protected mice from VSV challenge. These findings provide insights into the crosstalk between Tim-3 and other receptors in antiviral innate immunity and its related clinical significance.

Keywords: immunology; inflammation; mouse.

Cellular stress can induce cytoplasmic ribonucleoprotein complexes called stress granules that allow the cells to survive. Stress granules are also central to cellular responses to infections, in which they can act as platforms for viral sending or modulate innate immune signaling through pattern recognition receptors. However, the effect of innate immune signaling on stress granules is poorly understood. In this study, we report that prior induction of innate immune signaling through TLRs inhibited stress granule assembly in a TLR ligand dose-dependent manner in murine bone marrow-derived macrophages. Time course analysis suggests that TLR stimulation can reverse stress granule assembly even after it has begun. Additionally, both MYD88- and TRIF-mediated TLR signaling inhibited stress granule assembly in response to endoplasmic reticulum stress in bone marrow-derived macrophages and the chemotherapeutic drug oxaliplatin in murine B16 melanoma cells. This inhibition was not due to a decrease in expression of the critical stress granule proteins G3BP1 and DDX3X and was independent of IRAK1/4, JNK, ERK and P38 kinase activity but dependent on IKK complex kinase activity. Overall, we have identified the TLR-IKK complex signaling axis as a regulator of stress granule assembly-disassembly dynamics, highlighting cross-talk between processes that are critical in health and disease.

Translation arrest is a part of the cellular stress response that decreases energy consumption and enables rapid reprioritisation of gene expression. Often translation arrest leads to condensation of untranslated messenger ribonucleoproteins (mRNPs) into stress granules (SGs). Studies into mechanisms of SG formation and functions are complicated because various types of stress cause formation of SGs with different properties and composition. In this work we focused on the mechanism of SG formation triggered by UV damage. We demonstrate that UV-induced inhibition of translation does not involve inhibition of the mechanistic target of rapamycin (mTOR) signaling or dissociation of the 48S preinitiation complexes. The general control non-derepressible 2 (GCN2) kinase contributes to UV-induced SG formation, which is independent of the phosphorylation of the eukaryotic translation initiation factor 2α. Like many other types of SGs, condensation of UV-induced granules requires the Ras-GTPase-Activating Protein SH3-Domain-Binding Protein 1 (G3BP1). Our work reveals that in UV-treated cells the mechanisms of translation arrest and SG formation may be unlinked, resulting in SGs that do not contain the major type of polysome-free preinitiation complexes that accumulate in the cytoplasm.

Keywords: G3BP1; GCN2; MTOR; Stress granule; UVC.

Because of the lack of facile and accurate methods to track stress granule (SG) dynamics in live cells and in vivo, in-depth studies of the biological roles of this attractive membraneless organelle have been limited. Herein, we report the first small-molecule probe, TASG, for the selective, convenient and real-time monitoring of SGs. This novel molecule can simultaneously bind to SG RNAs, the core SG protein G3BP1, and their complexes, triggering a significant enhancement in fluorescence intensity, making TASG broadly applicable to SG imaging under various stress conditions in fixed and live cells, ex vivo and in vivo. Using TASG, the complicated endogenous SG dynamics were revealed in both live cells and C. elegans. Collectively, our work provides an ideal probe that has thus far been absent in the field of SG investigations. We anticipate that this powerful tool may create exciting opportunities to investigate the underlying roles of SGs in different organisms.

RNA-binding proteins (RBPs) can regulate more than a single aspect of RNA metabolism. We searched for such previously undiscovered multifunctionality within a set of 143 RBPs, by defining the predictive value of RBP abundance for the transcription and translation levels of known RBP target genes across 80 human hearts. This led us to newly associate 27 RBPs with cardiac translational regulation in vivo. Of these, 21 impacted both RNA expression and translation, albeit for virtually independent sets of target genes. We highlight a subset of these, including G3BP1, PUM1, UCHL5, and DDX3X, where dual regulation is achieved through differential affinity for target length, by which separate biological processes are controlled. Like the RNA helicase DDX3X, the known splicing factors EFTUD2 and PRPF8-all identified as multifunctional RBPs by our analysis-selectively influence target translation rates depending on 5' UTR structure. Our analyses identify dozens of RBPs as being multifunctional and pinpoint potential novel regulators of translation, postulating unanticipated complexity of protein-RNA interactions at consecutive stages of gene expression.

We report a new mechanism of androgen receptor (AR) mRNA regulation and cytoprotection in response to AR pathway inhibition (ARPI) stress in prostate cancer (PCA). AR mRNA translation is coordinately regulated by RNA binding proteins, YTHDF3 and G3BP1. Under ambient conditions m6A-modified AR mRNA is bound by YTHDF3 and translationally stimulated, while m6A-unmodified AR mRNA is bound by G3BP1 and translationally repressed. When AR-regulated PCA cell lines are subjected to ARPI stress, m6A-modified AR mRNA is recruited from actively translating polysomes (PSs) to RNA-protein stress granules (SGs), leading to reduced AR mRNA translation. After ARPI stress, m6A-modified AR mRNA liquid-liquid phase separated with YTHDF3, while m6A-unmodified AR mRNA phase separated with G3BP1. Accordingly, these AR mRNA messages form two distinct YTHDF3-enriched or G3BP1-enriched clusters in SGs. ARPI-induced SG formation is cell-protective, which when blocked by YTHDF3 or G3BP1 silencing increases PCA cell death in response to ARPI stress. Interestingly, AR mRNA silencing also delays ARPI stress-induced SG formation, highlighting its supportive role in triggering this stress response. Our results define a new mechanism for stress adaptive cell survival after ARPI stress involving SG-regulated translation of AR mRNA, mediated by m6A RNA modification and their respective regulatory proteins.

Chronic cellular stress associated with neurodegenerative disease can result in the persistence of stress granule (SG) structures, membraneless organelles that form in response to cellular stress. In Huntington's disease (HD), chronic expression of mutant huntingtin generates various forms of cellular stress, including activation of the unfolded protein response and oxidative stress. However, it has yet to be determined whether SGs are a feature of HD neuropathology. We examined the miRNA composition of extracellular vesicles (EVs) present in the cerebrospinal fluid (CSF) of HD patients and show that a subset of their target mRNAs were differentially expressed in the prefrontal cortex of HD patients. Of these targets, SG components were enriched, including the SG nucleating Ras GTPase-activating protein-binding protein 1 (G3BP1). We investigated localization and levels of G3BP1 and found a significant increase in the density of G3BP1-positive granules in the cortex and hippocampus of R6/2 transgenic mice and in the superior frontal cortex of HD patient brains. Intriguingly, we also observed that the SG-associated TAR DNA-Binding Protein-43 (TDP43), a nuclear RNA/DNA binding protein, was mislocalized to the cytoplasm of G3BP1-granule positive HD cortical neurons. These findings suggest that G3BP1 SG dynamics may play a role in the pathophysiology of HD.

Keywords: Neurodegeneration; Neuroscience.

Breast cancer heterogeneity has made it challenging to identify mechanisms critical to the initial stages of their genesis in vivo. Here, we sought to interrogate the role of YB-1 in newly arising human breast cancers as well as in established cell lines. In a first series of experiments, we found that short-hairpin RNA-mediated knockdown of YB-1 in MDA-MB-231 cells blocked both their local tumour-forming and lung-colonising activity in immunodeficient mice. Conversely, upregulated expression of YB-1 enhanced the poor in vivo tumorigenicity of T47D cells. We then found that YB-1 knockdown also inhibits the initial generation in mice of invasive ductal carcinomas and ductal carcinomas in situ from freshly isolated human mammary cells transduced, respectively, with KRAS^G12D or myristoylated-AKT1. Interestingly, increased expression of HIF1α and G3BP1, two YB-1 translational targets and elements of a stress-adaptive programme, mirrored the levels of YB-1 in both transformed primary and established MDA-MB-231 breast cancer cells.