The Role of Heat-Induced Stress Granules in the Blood-Testis Barrier of Mice.

Stress granules (SGs) are membraneless ribonucleoprotein (RNP)-based cellular foci formed in response to stress, facilitating cell survival by protecting against damage. Mammalian spermatogenesis should be maintained below body temperature for proper development, indicating its vulnerability to heat stress (HS). In this study, biotin tracer permeability assays showed that the inhibition of heat-induced SG assembly in the testis by 4-8 mg/kg cycloheximide significantly increased the percentage of seminiferous tubules with a damaged blood-testis barrier (BTB). Western blot results additionally revealed that the suppression of heat-induced SG assembly in Sertoli cell line, TM4 cells, by RNA inference of G3bp1/2 aggravated the decline in the BTB-related proteins ZO-1, ß-Catenin and Claudin-11, indicating that SGs could protect the BTB against damage caused by HS. The protein components that associate with SGs in Sertoli cells were isolated by sequential centrifugation and immunoprecipitation, and were identified by liquid chromatography with tandem mass spectrometry. Gene Ontology and KEGG pathway enrichment analysis revealed that their corresponding genes were mainly involved in pathways related to proteasomes, nucleotide excision repair, mismatch repair, and DNA replication. Furthermore, a new SG component, the ubiquitin associated protein 2 (UBAP2), was found to translocate to SGs upon HS in TM4 cells by immunofluorescence. Moreover, SG assembly was significantly diminished after UBAP2 knockdown by RNA inference during HS, suggesting the important role of UBAP2 in SG assembly. In addition, UBAP2 knockdown reduced the expression of ZO-1, ß-Catenin and Claudin-11, which implied its potential role in the function of the BTB. Overall, our study demonstrated the role of SGs in maintaining BTB functions during HS and identified a new component implicated in SG formation in Sertoli cells. These findings not only offer novel insights into the biological functions of SGs and the molecular mechanism of low fertility in males in summer, but also potentially provide an experimental basis for male fertility therapies.

Shining light on DHX9: UV-induced stress granules illuminate protective mechanisms for daughter cell resilience.

In a recent article in Cell, Zhou et al. investigate the origins, composition, and biological consequences of UV-induced stress granules. They find that UV-induced stress granules are triggered by the formation of RNA-protein crosslinks, uniquely contain DHX9 as a marker, form during mitosis independently of translation repression, and are enriched in intron-containing RNAs and splicing factors. Moreover, UV-induced granules contain double-stranded RNA (dsRNA) and trigger a dsRNA response. This work identifies a mechanism for resolving UV-damaged RNA and broadens the types of cytosolic "stress granules" that form.

Apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) promotes stress granule formation via YBX1 phosphorylation in ovarian cancer.

APE1 is an essential gene involved in DNA damage repair, the redox regulation of transcriptional factors (TFs) and RNA processing. APE1 overexpression is common in cancers and correlates with poor patient survival. Stress granules (SGs) are phase-separated cytoplasmic assemblies that cells form in response to environmental stresses. Precise regulation of SGs is pivotal to cell survival, whereas their dysregulation is increasingly linked to diseases. Whether APE1 engages in modulating SG dynamics is worthy of investigation. In this study, we demonstrate that APE1 colocalizes with SGs and promotes their formation. Through phosphoproteome profiling, we discover that APE1 significantly alters the phosphorylation landscape of ovarian cancer cells, particularly the phosphoprofile of SG proteins. Notably, APE1 promotes the phosphorylation of Y-Box binding protein 1 (YBX1) at S174 and S176, leading to enhanced SG formation and cell survival. Moreover, expression of the phosphomutant YBX1 S174/176E mimicking hyperphosphorylation in APE1-knockdown cells recovered the impaired SG formation. These findings shed light on the functional importance of APE1 in SG regulation and highlight the importance of YBX1 phosphorylation in SG dynamics.

DDX6 modulates P-body and stress granule assembly, composition, and docking.

Stress granules and P-bodies are ribonucleoprotein (RNP) granules that accumulate during the stress response due to the condensation of untranslating mRNPs. Stress granules form in part by intermolecular RNA-RNA interactions and can be limited by components of the RNA chaperone network, which inhibits RNA-driven aggregation. Herein, we demonstrate that the DEAD-box helicase DDX6, a P-body component, can also limit the formation of stress granules, independent of the formation of P-bodies. In an ATPase, RNA-binding dependent manner, DDX6 limits the partitioning of itself and other RNPs into stress granules. When P-bodies are limited, proteins that normally partition between stress granules and P-bodies show increased accumulation within stress granules. Moreover, we show that loss of DDX6, 4E-T, and DCP1A increases P-body docking with stress granules, which depends on CNOT1 and PAT1B. Taken together, these observations identify a new role for DDX6 in limiting stress granules and demonstrate that P-body components can influence stress granule composition and docking with P-bodies.

RNA damage compartmentalization by DHX9 stress granules.

Biomolecules incur damage during stress conditions, and damage partitioning represents a vital survival strategy for cells. Here, we identified a distinct stress granule (SG), marked by dsRNA helicase DHX9, which compartmentalizes ultraviolet (UV)-induced RNA, but not DNA, damage. Our FANCI technology revealed that DHX9 SGs are enriched in damaged intron RNA, in contrast to classical SGs that are composed of mature mRNA. UV exposure causes RNA crosslinking damage, impedes intron splicing and decay, and triggers DHX9 SGs within daughter cells. DHX9 SGs promote cell survival and induce dsRNA-related immune response and translation shutdown, differentiating them from classical SGs that assemble downstream of translation arrest. DHX9 modulates dsRNA abundance in the DHX9 SGs and promotes cell viability. Autophagy receptor p62 is activated and important for DHX9 SG disassembly. Our findings establish non-canonical DHX9 SGs as a dedicated non-membrane-bound cytoplasmic compartment that safeguards daughter cells from parental RNA damage.

hnRNPA2B1 represses the disassembly of arsenite-induced stress granules and is essential for male fertility.

Although the composition and assembly of stress granules (SGs) are well understood, the molecular mechanisms underlying SG disassembly remain unclear. Here, we identify that heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) is associated with SGs and that its absence specifically enhances the disassembly of arsenite-induced SGs depending on the ubiquitination-proteasome system but not the autophagy pathway. hnRNPA2B1 interacts with many core SG proteins, including G3BP1, G3BP2, USP10, and Caprin-1; USP10 can deubiquitinate G3BP1; and hnRNPA2B1 depletion attenuates the G3BP1-USP10/Caprin-1 interaction but elevates the G3BP1 ubiquitination level under arsenite treatment. Moreover, the disease-causing mutation FUSR521C also disassembles faster from SGs in HNRNPA2B1 mutant cells. Furthermore, knockout of hnRNPA2B1 in mice leads to Sertoli cell-only syndrome (SCOS), causing complete male infertility. Consistent with this, arsenite-induced SGs disassemble faster in Hnrnpa2b1 knockout (KO) mouse Sertoli cells as well. These findings reveal the essential roles of hnRNPA2B1 in regulating SG disassembly and male mouse fertility.

Shigella induces stress granule formation by ADP-riboxanation of the eIF3 complex.

Under stress conditions, translationally stalled mRNA and associated proteins undergo liquid-liquid phase separation and condense into cytoplasmic foci called stress granules (SGs). Many viruses hijack SGs for their pathogenesis; however, whether pathogenic bacteria also exploit this pathway remains unknown. Here, we report that members of the OspC family of Shigella flexneri induce SG formation in infected cells. Mechanistically, the OspC effectors target multiple subunits of the host translation initiation factor 3 complex by ADP-riboxanation. The modification of eIF3 leads to translational arrest and thus the formation of SGs. Furthermore, OspC-mediated SGs are beneficial for S. flexneri replication within infected host cells, and bacterial strains unable to induce SGs are attenuated for virulence in a murine model of infection. Our findings reveal a mechanism by which bacterial pathogens induce SG assembly by inactivating host translational machinery and promote bacterial proliferation in host cells.

Escherichia coli-Induced cGLIS3-Mediated Stress Granules Activate the NF-κB Pathway to Promote Intrahepatic Cholangiocarcinoma Progression.

Patients with concurrent intrahepatic cholangiocarcinoma (ICC) and hepatolithiasis generally have poor prognoses. Hepatolithiasis is once considered the primary cause of ICC, although recent insights indicate that bacteria in the occurrence of hepatolithiasis can promote the progression of ICC. By constructing in vitro and in vivo ICC models and patient-derived organoids (PDOs), it is shown that Escherichia coli induces the production of a novel RNA, circGLIS3 (cGLIS3), which promotes tumor growth. cGLIS3 binds to hnRNPA1 and G3BP1, resulting in the assembly of stress granules (SGs) and suppression of hnRNPA1 and G3BP1 ubiquitination. Consequently, the IKKα mRNA is blocked in SGs, decreasing the production of IKKα and activating the NF-κB pathway, which finally results in chemoresistance and produces metastatic phenotypes of ICC. This study shows that a combination of Icaritin (ICA) and gemcitabine plus cisplatin (GP) chemotherapy can be a promising treatment strategy for ICC.

Amperometry and Electron Microscopy show Stress Granules Induce Homotypic Fusion of Catecholamine Vesicles.

An overreactive stress granule (SG) pathway and long-lived, stable SGs formation are thought to participate in the progress of neurodegenerative diseases (NDs). To understand if and how SGs contribute to disorders of neurotransmitter release in NDs, we examined the interaction between extracellular isolated SGs and vesicles. Amperometry shows that the vesicular content increases and dynamics of vesicle opening slow down after vesicles are treated with SGs, suggesting larger vesicles are formed. Data from transmission electron microscopy (TEM) clearly shows that a portion of large dense-core vesicles (LDCVs) with double/multiple cores appear, thus confirming that SGs induce homotypic fusion between LDCVs. This might be a protective step to help cells to survive following high oxidative stress. A hypothetical mechanism is proposed whereby enriched mRNA or protein in the shell of SGs is likely to bind intrinsically disordered protein (IDP) regions of vesicle associated membrane protein (VAMP) driving a disrupted membrane between two closely buddled vesicles to fuse with each other to form double-core vesicles. Our results show that SGs induce homotypic fusion of LDCVs, providing better understanding of how SGs intervene in pathological processes and opening a new direction to investigations of SGs involved neurodegenerative disease.

N4-acetylcytidine (ac4C) promotes mRNA localization to stress granules.

Stress granules are an integral part of the stress response that are formed from non-translating mRNAs aggregated with proteins. While much is known about stress granules, the factors that drive their mRNA localization are incompletely described. Modification of mRNA can alter the properties of the nucleobases and affect processes such as translation, splicing and localization of individual transcripts. Here, we show that the RNA modification N4-acetylcytidine (ac4C) on mRNA associates with transcripts enriched in stress granules and that stress granule localized transcripts with ac4C are specifically translationally regulated. We also show that ac4C on mRNA can mediate localization of the protein NOP58 to stress granules. Our results suggest that acetylation of mRNA regulates localization of both stress-sensitive transcripts and RNA-binding proteins to stress granules and adds to our understanding of the molecular mechanisms responsible for stress granule formation.

HuR-positive stress granules: Potential targets for age-related osteoporosis.

Aging impairs osteoblast function and bone turnover, resulting in age-related bone degeneration. Stress granules (SGs) are membrane-less organelles that assemble in response to stress via the recruitment of RNA-binding proteins (RBPs), and have emerged as a novel mechanism in age-related diseases. Here, we identified HuR as a bone-related RBP that aggregated into SGs and facilitated osteogenesis during aging. HuR-positive SG formation increased during osteoblast differentiation, and HuR overexpression mitigated the reduction in SG formation observed in senescent osteoblasts. Moreover, HuR positively regulated the mRNA stability and expression of its target ß-catenin by binding and recruiting ß-catenin into SGs. As a potential therapeutic target, HuR activator apigenin (API) enhanced its expression and thus aided osteoblasts differentiation. API treatment increased HuR nuclear export, enhanced the recruitment of ß-catenin into HuR-positive SGs, facilitated ß-catenin nuclear translocation, and contributed osteogenesis. Our findings highlight the roles of HuR and its SGs in promoting osteogenesis during skeletal aging and lay the groundwork for novel therapeutic strategies against age-related skeletal disorders.

Targeting NUPR1-dependent stress granules formation to induce synthetic lethality in KrasG12D-driven tumors.

We find that NUPR1, a stress-associated intrinsically disordered protein, induced droplet formation via liquid-liquid phase separation (LLPS). NUPR1-driven LLPS was crucial for the creation of NUPR1-dependent stress granules (SGs) in pancreatic cancer cells since genetic or pharmacological inhibition by ZZW-115 of NUPR1 activity impeded SGs formation. The KrasG12D mutation induced oncogenic stress, NUPR1 overexpression, and promoted SGs development. Notably, enforced NUPR1 expression induced SGs formation independently of mutated KrasG12D. Mechanistically, KrasG12D expression strengthened sensitivity to NUPR1 inactivation, inducing cell death, activating caspase 3 and releasing LDH. Remarkably, ZZW-115-mediated SG-formation inhibition hampered the development of pancreatic intraepithelial neoplasia (PanINs) in Pdx1-cre;LSL-KrasG12D (KC) mice. ZZW-115-treatment of KC mice triggered caspase 3 activation, DNA fragmentation, and formation of the apoptotic bodies, leading to cell death, specifically in KrasG12D-expressing cells. We further demonstrated that, in developed PanINs, short-term ZZW-115 treatment prevented NUPR1-associated SGs presence. Lastly, a four-week ZZW-115 treatment significantly reduced the number and size of PanINs in KC mice. This study proposes that targeting NUPR1-dependent SGs formation could be a therapeutic approach to induce cell death in KrasG12D-dependent tumors.

Dysregulation of stress granule dynamics by DCTN1 deficiency exacerbates TDP-43 pathology in Drosophila models of ALS/FTD.

The abnormal aggregation of TDP-43 into cytoplasmic inclusions in affected neurons is a major pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although TDP-43 is aberrantly accumulated in the neurons of most patients with sporadic ALS/FTD and other TDP-43 proteinopathies, how TDP-43 forms cytoplasmic aggregates remains unknown. In this study, we show that a deficiency in DCTN1, a subunit of the microtubule-associated motor protein complex dynactin, perturbs the dynamics of stress granules and drives the formation of TDP-43 cytoplasmic aggregation in cultured cells, leading to the exacerbation of TDP-43 pathology and neurodegeneration in vivo. We demonstrated using a Drosophila model of ALS/FTD that genetic knockdown of DCTN1 accelerates the formation of ubiquitin-positive cytoplasmic inclusions of TDP-43. Knockdown of components of other microtubule-associated motor protein complexes, including dynein and kinesin, also increased the formation of TDP-43 inclusions, indicating that intracellular transport along microtubules plays a key role in TDP-43 pathology. Notably, DCTN1 knockdown delayed the disassembly of stress granules in stressed cells, leading to an increase in the formation of pathological cytoplasmic inclusions of TDP-43. Our results indicate that a deficiency in DCTN1, as well as disruption of intracellular transport along microtubules, is a modifier that drives the formation of TDP-43 pathology through the dysregulation of stress granule dynamics.

Regulation of stress granule formation in human oligodendrocytes.

Oligodendrocyte (OL) injury and subsequent loss is a pathologic hallmark of multiple sclerosis (MS). Stress granules (SGs) are membrane-less organelles containing mRNAs stalled in translation and considered as participants of the cellular response to stress. Here we show SGs in OLs in active and inactive areas of MS lesions as well as in normal-appearing white matter. In cultures of primary human adult brain derived OLs, metabolic stress conditions induce transient SG formation in these cells. Combining pro-inflammatory cytokines, which alone do not induce SG formation, with metabolic stress results in persistence of SGs. Unlike sodium arsenite, metabolic stress induced SG formation is not blocked by the integrated stress response inhibitor. Glycolytic inhibition also induces persistent SGs indicating the dependence of SG formation and disassembly on the energetic glycolytic properties of human OLs. We conclude that SG persistence in OLs in MS reflects their response to a combination of metabolic stress and pro-inflammatory conditions.

YBX1 Underwent Phase Separation into Stress Granules Stimulated by Ionizing Radiation.

Stress granules (SGs) are formed through liquid-liquid phase separation (LLPS), in response to external stimuli. YBX1, an integral component of SGs, plays a crucial role in tumor progression and cellular stress response. This study aims to elucidate the mechanisms and specific biological implications of YBX1 in SG formation, along with the identification of key regions and interacting proteins. Our observations indicate that YBX1 rapidly undergoes liquid-liquid phase separation, leading to SG formation in response to 8 Gy X-ray irradiation within 1 h, with SGs reverting to their original state after 5 h. There was a potential interaction between ATXN2L and YBX1, persisting YBX1 within the SGs. Our data suggested a potential interaction between ATXN2L and YBX1, and it remained associated with YBX1 within the SGs. Furthermore, our subsequent studies demonstrate that targeting ATXN2L can diminish the recruitment of YBX1 to stress granules (SGs), consequently enhancing the radiosensitivity of HeLa cells.

Stress granule and P-body clearance: Seeking coherence in acts of disappearance.

Stress granules and P-bodies are conserved cytoplasmic biomolecular condensates whose assembly and composition are well documented, but whose clearance mechanisms remain controversial or poorly described. Such understanding could provide new insight into how cells regulate biomolecular condensate formation and function, and identify therapeutic strategies in disease states where aberrant persistence of stress granules in particular is implicated. Here, I review and compare the contributions of chaperones, the cytoskeleton, post-translational modifications, RNA helicases, granulophagy and the proteasome to stress granule and P-body clearance. Additionally, I highlight the potentially vital role of RNA regulation, cellular energy, and changes in the interaction networks of stress granules and P-bodies as means of eliciting clearance. Finally, I discuss evidence for interplay of distinct clearance mechanisms, suggest future experimental directions, and suggest a simple working model of stress granule clearance.

Muscone alleviates neuronal injury via increasing stress granules formation and reducing apoptosis in acute ischemic stroke.

As the main bioactive component of musk, muscone has been reported to have marked protective effects in treating acute ischemic stroke (AIS). However, the specific anti-stroke mechanism of muscone still needs further research. In the current investigation, the PC12 cells OGD/R and the rat transient MCAO/R models were utilized as the AIS models. Serum hepatic and renal functional indexes (ALT, AST, BUN, and Cr) and cell viability were determined to select the appropriate muscone concentrations for in vitro and in vivo experiments. TTC, Hematoxylin and eosin (H&E), and Live/Dead staining were utilized to evaluate the protective effects of muscone in injured tissues and cells. Western blotting analysis, TUNEL staining, propidium iodide, and annexin V staining were applied to detect the anti-apoptotic effect of muscone. Double-label immunofluorescence staining of T-cell intracellular antigen-1 (TIA1) and Ras-GAP SH3 domain-binding protein 1 (G3BP1) was performed to observe whether muscone regulated the SG formation level. Molecular docking, TIA1 silencing and TIA1 overexpression experiments were employed to investigate the molecular mechanism underlying the regulation of SG formation by muscone. The 2, 3, 5-Triphenyl-tetrazolium chloride (TTC) staining and live/dead staining showed the AIS injury level of MCAO/R rat and the OGD/R PC12 cells were attenuated by muscone administration. The muscone significantly minimized the apoptosis rate in MCAO/R rats and OGD/R PC12 cells following flow cytometry analysis, western blotting analysis, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The double-label immunofluorescence staining data revealed that muscone promoted the SG formation level in OGD/R PC12 cells and the cortex MCAO/R rats. The results of molecular docking, TIA1 silencing and TIA1 overexpression experiments revealed that muscone could bind to TIA1 protein and regulate its expression level, thereby promoting the formation of stress granules and exerting a protective effect against AIS injury. This study indicated that the significant protective effect of muscone in reducing apoptosis levels might be via promoting SG formation under AIS conditions. This study further explores the therapeutic effect and anti-apoptosis mechanism of muscone in AIS, which may provide a potential candidate drug for the clinical treatment of AIS injury.

Immunofluorescence Combined with Single-Molecule RNA Fluorescence In Situ Hybridization for Concurrent Detection of Proteins and Transcripts in Stress Granules.

Immunofluorescence (IF) microscopy is arguably one of the most commonly used methods for studying structure and composition of stress granules (SGs). While in most cases standard IF protocols are sufficient to visualize protein components of SGs, concurrent detection of proteins and transcripts in stress granules requires more sophisticated and problematic approaches. Here we present a well-established, simple, robust, and fluorescent protein-compatible method for simultaneous detection of proteins and transcripts in individual stress granules using combination of IF and single-molecule RNA fluorescence in situ hybridization (smRNA FISH).