ABSTRACT
Negative-strand RNA viruses form cytoplasmic inclusion bodies (IBs) representing virus replication foci through phase separation or biomolecular condensation of viral and cellular proteins, as a hallmark of their infection. Alternatively, mammalian cells form stalled mRNA containing antiviral stress granules (SGs), as a consequence of phosphorylation of eukaryotic initiation factor 2α (eIF2α) through condensation of several RNA-binding proteins including TIA-1. Whether and how Chandipura virus (CHPV), an emerging human pathogen causing influenza-like illness, coma and death, forms IBs and evades antiviral SGs remain unknown. By confocal imaging on CHPV-infected Vero-E6 cells, we found that CHPV infection does not induce formation of distinct canonical SGs. Instead, CHPV proteins condense and co-localize together with SG proteins to form heterogeneous IBs, which ensued independent of the activation of eIF2α and eIF2α kinase, protein kinase R (PKR). Interestingly, siRNA-mediated depletion of PKR or TIA-1 significantly decreased viral transcription and virion production. Moreover, CHPV infection also caused condensation and recruitment of PKR to IBs. Compared to SGs, IBs exhibited significant rapidity in disassembly dynamics. Altogether, our study demonstrating that CHPV replication co-optimizes with SG proteins and revealing an unprecedented proviral role of TIA-1/PKR may have implications in understanding the mechanisms regulating CHPV-IB formation and designing antiviral therapeutics. Importance: CHPV is an emerging tropical pathogen reported to cause acute influenza-like illness and encephalitis in children with a very high mortality rate of ~70%. Lack of vaccines and an effective therapy against CHPV makes it a potent pathogen for causing an epidemic in tropical parts of globe. Given these forewarnings, it is of paramount importance that CHPV biology must be understood comprehensively. Targeting of host factors offers several advantages over targeting the viral components due to the generally higher mutation rate in the viral genome. In this study, we aimed at understanding the role of SGs forming cellular RNA-binding proteins in CHPV replication. Our study helps understand participation of cellular factors in CHPV replication and could help develop effective therapeutics against the virus.
Subject(s)
Inclusion Bodies, Viral , T-Cell Intracellular Antigen-1 , Virus Replication , eIF-2 Kinase , eIF-2 Kinase/metabolism , eIF-2 Kinase/genetics , Animals , T-Cell Intracellular Antigen-1/metabolism , T-Cell Intracellular Antigen-1/genetics , Chlorocebus aethiops , Vero Cells , Inclusion Bodies, Viral/metabolism , Humans , Stress Granules/metabolism , Inclusion Bodies/metabolism , Host-Pathogen Interactions , Cytoplasmic Granules/metabolism , Viral Proteins/metabolism , Viral Proteins/genetics , Phase SeparationABSTRACT
Stress triggers the formation of two distinct cytoplasmic biomolecular condensates: stress granules (SGs) and processing bodies (PBs), both of which may contribute to stress-responsive translation regulation. Though PBs can be present constitutively, stress can increase their number and size and lead to their interaction with stress-induced SGs. The mechanism of such interaction, however, is largely unknown. Formation of canonical SGs requires the RNA binding protein Ubiquitin-Associated Protein 2-Like (UBAP2L), which is a central SG node protein in the RNA-protein interaction network of SGs and PBs. UBAP2L binds to the essential SG and PB proteins G3BP and DDX6, respectively. Research on UBAP2L has mostly focused on its role in SGs, but not its connection to PBs. We find that UBAP2L is not solely an SG protein but also localizes to PBs in certain conditions, contributes to PB biogenesis and SG-PB interactions, and can nucleate hybrid granules containing SG and PB components in cells. These findings inform a new model for SG and PB formation in the context of UBAP2L's role.
Subject(s)
RNA Helicases , RNA Recognition Motif Proteins , Stress Granules , Humans , Stress Granules/metabolism , Stress Granules/genetics , RNA Helicases/metabolism , RNA Helicases/genetics , RNA Recognition Motif Proteins/metabolism , RNA Recognition Motif Proteins/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Processing Bodies/metabolism , Processing Bodies/genetics , DEAD-box RNA Helicases/metabolism , DEAD-box RNA Helicases/genetics , Cytoplasmic Granules/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , HeLa Cells , DNA Helicases/metabolism , DNA Helicases/genetics , HEK293 Cells , Protein Binding , Carrier Proteins/metabolism , Carrier Proteins/genetics , Proto-Oncogene ProteinsABSTRACT
Neurologic manifestations are an immediate consequence of SARS-CoV-2 infection, the etiologic agent of COVID-19, which, however, may also trigger long-term neurological effects. Notably, COVID-19 patients with neurological symptoms show elevated levels of biomarkers associated with brain injury, including Tau proteins linked to Alzheimer's pathology. Studies in brain organoids revealed that SARS-CoV-2 alters the phosphorylation and distribution of Tau in infected neurons, but the mechanisms are currently unknown. We hypothesize that these pathological changes are due to the recruitment of Tau into stress granules (SGs) operated by the nucleocapsid protein (NCAP) of SARS-CoV-2. To test this hypothesis, we investigated whether NCAP interacts with Tau and localizes to SGs in hippocampal neurons in vitro and in vivo. Mechanistically, we tested whether SUMOylation, a posttranslational modification of NCAP and Tau, modulates their distribution in SGs and their pathological interaction. We found that NCAP and Tau colocalize and physically interact. We also found that NCAP induces hyperphosphorylation of Tau and causes cognitive impairment in mice infected with NCAP in their hippocampus. Finally, we found that SUMOylation modulates NCAP SG formation in vitro and cognitive performance in infected mice. Our data demonstrate that NCAP induces Tau pathological changes both in vitro and in vivo. Moreover, we demonstrate that SUMO2 ameliorates NCAP-induced Tau pathology, highlighting the importance of the SUMOylation pathway as a target of intervention against neurotoxic insults, such as Tau oligomers and viral infection.
Subject(s)
COVID-19 , Coronavirus Nucleocapsid Proteins , Hippocampus , Neurons , SARS-CoV-2 , Sumoylation , tau Proteins , tau Proteins/metabolism , Animals , Mice , Humans , Hippocampus/metabolism , Hippocampus/pathology , COVID-19/metabolism , COVID-19/pathology , COVID-19/virology , SARS-CoV-2/pathogenicity , SARS-CoV-2/metabolism , Phosphorylation , Coronavirus Nucleocapsid Proteins/metabolism , Neurons/metabolism , Neurons/pathology , Neurons/virology , Small Ubiquitin-Related Modifier Proteins/metabolism , Stress Granules/metabolism , Mice, Inbred C57BL , Phosphoproteins/metabolism , Male , Nucleocapsid Proteins/metabolism , Cognitive Dysfunction/metabolism , Cognitive Dysfunction/pathology , Cognitive Dysfunction/virologyABSTRACT
The cell interior is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cellular physiology. Cellular stress responses almost universally lead to inhibition of translation, resulting in polysome collapse and release of mRNA. The released mRNA molecules condense with RNA-binding proteins to form ribonucleoprotein (RNP) condensates known as processing bodies and stress granules. Here, we show that polysome collapse and condensation of RNA transiently fluidize the cytoplasm, and coarse-grained molecular dynamic simulations support this as a minimal mechanism for the observed biophysical changes. Increased mesoscale diffusivity correlates with the efficient formation of quality control bodies (Q-bodies), membraneless organelles that compartmentalize misfolded peptides during stress. Synthetic, light-induced RNA condensation also fluidizes the cytoplasm. Together, our study reveals a functional role for stress-induced translation inhibition and formation of RNP condensates in modulating the physical properties of the cytoplasm to enable efficient response of cells to stress conditions.
Subject(s)
Cytoplasm , Polyribosomes , Ribonucleoproteins , Polyribosomes/metabolism , Cytoplasm/metabolism , Humans , Ribonucleoproteins/metabolism , Ribonucleoproteins/genetics , Molecular Dynamics Simulation , RNA, Messenger/metabolism , RNA, Messenger/genetics , Protein Biosynthesis , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Biomolecular Condensates/metabolism , Stress Granules/metabolism , Stress Granules/geneticsABSTRACT
Stress granules (SGs), formed by untranslated messenger ribonucleoproteins (mRNPs) during cellular stress in eukaryotes, have been linked to flavivirus interference without clear understanding. This study reveals the role of Zika virus (ZIKV) NS2B as a scaffold protein mediating interaction between protein phosphatase 1α (PP1α) and eukaryotic initiation factor 2α (eIF2α). This interaction promotes eIF2α dephosphorylation by PP1α, inhibiting SG formation. The NS2B-PP1α complex exhibits remarkable stability, resisting ubiquitin-induced degradation and amplifying eIF2α dephosphorylation, thus promoting ZIKV replication. In contrast, the NS2BV35A mutant, interacting exclusively with eIF2α, fails to inhibit SG formation, resulting in reduced viral replication and diminished impact on brain organoid growth. These findings reveal PP1α's dual role in ZIKV infection, inducing interferon production as an antiviral factor and suppressing SG formation as a viral promoter. Moreover, we found that NS2B also serves as a versatile mechanism employed by flaviviruses to counter host antiviral defenses, primarily by broadly inhibiting SG formation. This research advances our comprehension of the complex interplay in flavivirus-host interactions, offering potential for innovative therapeutic strategies against flavivirus infections.
Subject(s)
Eukaryotic Initiation Factor-2 , Protein Phosphatase 1 , Stress Granules , Viral Nonstructural Proteins , Virus Replication , Zika Virus Infection , Zika Virus , Zika Virus/physiology , Virus Replication/physiology , Humans , Zika Virus Infection/virology , Zika Virus Infection/metabolism , Viral Nonstructural Proteins/metabolism , Viral Nonstructural Proteins/genetics , Protein Phosphatase 1/metabolism , Eukaryotic Initiation Factor-2/metabolism , Stress Granules/metabolism , AnimalsABSTRACT
METTL3 is the catalytic subunit of the methyltransferase complex, which mediates m6A modification to regulate gene expression. In addition, METTL3 regulates transcription in an enzymatic activity-independent manner by driving changes in high-order chromatin structure. However, how these functions of the methyltransferase complex are coordinated remains unknown. Here we show that the methyltransferase complex coordinates its enzymatic activity-dependent and independent functions to regulate cellular senescence, a state of stable cell growth arrest. Specifically, METTL3-mediated chromatin loops induce Hexokinase 2 expression through the three-dimensional chromatin organization during senescence. Elevated Hexokinase 2 expression subsequently promotes liquid-liquid phase separation, manifesting as stress granule phase separation, by driving metabolic reprogramming. This correlates with an impairment of translation of cell-cycle related mRNAs harboring polymethylated m6A sites. In summary, our results report a coordination of m6A-dependent and -independent function of the methyltransferase complex in regulating senescence through phase separation driven by metabolic reprogramming.
Subject(s)
Cellular Senescence , Chromatin , Methyltransferases , Stress Granules , Methyltransferases/metabolism , Methyltransferases/genetics , Chromatin/metabolism , Humans , Stress Granules/metabolism , Stress Granules/genetics , Hexokinase/metabolism , Hexokinase/genetics , RNA, Messenger/metabolism , RNA, Messenger/genetics , Adenosine/metabolism , Adenosine/analogs & derivatives , HEK293 Cells , Metabolic Reprogramming , Phase SeparationABSTRACT
Some chemotherapy drugs modulate the formation of stress granules (SGs), which are RNA-containing cytoplasmic foci contributing to stress response pathways. How SGs mechanistically contribute to pro-survival or pro-apoptotic functions must be better defined. The chemotherapy drug lomustine promotes SG formation by activating the stress-sensing eIF2α kinase HRI (encoded by the EIF2AK1 gene). Here, we applied a DNA microarray-based transcriptome analysis to determine the genes modulated by lomustine-induced stress and suggest roles for SGs in this process. We found that the expression of the pro-apoptotic EGR1 gene was specifically regulated in cells upon lomustine treatment. The appearance of EGR1-encoding mRNA in SGs correlated with a decrease in EGR1 mRNA translation. Specifically, EGR1 mRNA was sequestered to SGs upon lomustine treatment, probably preventing its ribosome translation and consequently limiting the degree of apoptosis. Our data support the model where SGs can selectively sequester specific mRNAs in a stress-specific manner, modulate their availability for translation, and thus determine the fate of a stressed cell.
Subject(s)
Early Growth Response Protein 1 , Lomustine , RNA, Messenger , Humans , RNA, Messenger/metabolism , RNA, Messenger/genetics , Early Growth Response Protein 1/metabolism , Early Growth Response Protein 1/genetics , Lomustine/pharmacology , Stress Granules/metabolism , Stress Granules/genetics , Apoptosis/drug effects , Antineoplastic Agents, Alkylating/pharmacologyABSTRACT
(1) Background: Stress granules (SGs) are cytoplasmic protein-RNA condensates that assemble in response to various insults. SG production is driven by signaling pathways that are relevant to human disease. Compounds that modulate SG characteristics are therefore of clinical interest. Pifithrin-µ is a candidate anti-tumor agent that inhibits members of the hsp70 chaperone family. While hsp70s are required for granulostasis, the impact of pifithrin-µ on SG formation is unknown. (2) Methods: Using HeLa cells as model system, cell-based assays evaluated the effects of pifithrin-µ on cell viability. Quantitative Western blotting assessed cell signaling events and SG proteins. Confocal microscopy combined with quantitative image analyses examined multiple SG parameters. (3) Results: Pifithrin-µ induced bona fide SGs in the absence of exogenous stress. These SGs were dynamic; their properties were determined by the duration of pifithrin-µ treatment. The phosphorylation of eIF2α was mandatory to generate SGs upon pifithrin-µ exposure. Moreover, the formation of pifithrin-µ SGs was accompanied by profound changes in cell signaling. Pifithrin-µ reduced the activation of 5'-AMP-activated protein kinase, whereas the pro-survival protein kinase Akt was activated. Long-term pifithrin-µ treatment caused a marked loss of cell viability. (4) Conclusions: Our study identified stress-related changes in cellular homeostasis that are elicited by pifithrin-µ. These insights are important knowledge for the appropriate therapeutic use of pifithrin-µ and related compounds.
Subject(s)
Cell Survival , Signal Transduction , Stress Granules , Humans , Cell Survival/drug effects , Signal Transduction/drug effects , HeLa Cells , Stress Granules/metabolism , Phosphorylation/drug effects , Toluene/analogs & derivatives , Toluene/pharmacology , Eukaryotic Initiation Factor-2/metabolism , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease due to gradual motoneurons (MN) degeneration. Among the processes associated to ALS pathogenesis, there is the formation of cytoplasmic inclusions produced by aggregation of mutant proteins, among which the RNA binding protein FUS. Here we show that, in neuronal cells and in iPSC-derived MN expressing mutant FUS, such inclusions are significantly reduced in number and dissolve faster when the RNA m6A content is diminished. Interestingly, stress granules formed in ALS conditions showed a distinctive transcriptome with respect to control cells, which reverted to similar to control after m6A downregulation. Notably, cells expressing mutant FUS were characterized by higher m6A levels suggesting a possible link between m6A homeostasis and pathological aggregates. Finally, we show that FUS inclusions are reduced also in patient-derived fibroblasts treated with STM-2457, an inhibitor of METTL3 activity, paving the way for its possible use for counteracting aggregate formation in ALS.
Subject(s)
Amyotrophic Lateral Sclerosis , Induced Pluripotent Stem Cells , Motor Neurons , RNA-Binding Protein FUS , RNA-Binding Protein FUS/metabolism , RNA-Binding Protein FUS/genetics , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Humans , Motor Neurons/metabolism , Motor Neurons/pathology , Induced Pluripotent Stem Cells/metabolism , Cytoplasmic Granules/metabolism , Fibroblasts/metabolism , Adenosine/metabolism , Adenosine/analogs & derivatives , Methyltransferases/metabolism , Methyltransferases/genetics , Mutation , Inclusion Bodies/metabolism , Stress Granules/metabolism , TranscriptomeABSTRACT
The model of RNA stability has undergone a transformative shift with the revelation of a cytoplasmic capping activity that means a subset of transcripts are recapped autonomously of their nuclear counterparts. The present study demonstrates nucleo-cytoplasmic shuttling of the mRNA-capping enzyme (CE, also known as RNA guanylyltransferase and 5'-phosphatase; RNGTT), traditionally acknowledged for its nuclear localization and functions, elucidating its contribution to cytoplasmic capping activities. A unique nuclear export sequence in CE mediates XPO1-dependent nuclear export of CE. Notably, during sodium arsenite-induced oxidative stress, cytoplasmic CE (cCE) congregates within stress granules (SGs). Through an integrated approach involving molecular docking and subsequent co-immunoprecipitation, we identify eIF3b, a constituent of SGs, as an interactive associate of CE, implying that it has a potential role in guiding cCE to SGs. We measured the cap status of specific mRNA transcripts from U2OS cells that were non-stressed, stressed and recovered from stress, which indicated that cCE-target transcripts lost their caps during stress but remarkably regained cap stability during the recovery phase. This comprehensive study thus uncovers a novel facet of cytoplasmic CE, which facilitates cellular recovery from stress by maintaining cap homeostasis of target mRNAs.
Subject(s)
Cytoplasm , Homeostasis , RNA, Messenger , Stress Granules , Humans , RNA, Messenger/metabolism , RNA, Messenger/genetics , Stress Granules/metabolism , Cytoplasm/metabolism , RNA Caps/metabolism , Arsenites/pharmacology , Oxidative Stress , Active Transport, Cell Nucleus , RNA Nucleotidyltransferases/metabolism , RNA Nucleotidyltransferases/genetics , Sodium Compounds/pharmacology , Exportin 1 Protein , Karyopherins/metabolism , Karyopherins/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Cytoplasmic Granules/metabolism , RNA Stability , Cell Nucleus/metabolism , Cell Line, Tumor , NucleotidyltransferasesABSTRACT
Stress granules (SGs) are induced by various environmental stressors, resulting in their compositional and functional heterogeneity. SGs play a crucial role in the antiviral process, owing to their potent translational repressive effects and ability to trigger signal transduction; however, it is poorly understood how these antiviral SGs differ from SGs induced by other environmental stressors. Here we identify that TRIM25, a known driver of the ubiquitination-dependent antiviral innate immune response, is a potent and critical marker of the antiviral SGs. TRIM25 undergoes liquid-liquid phase separation (LLPS) and co-condenses with the SG core protein G3BP1 in a dsRNA-dependent manner. The co-condensation of TRIM25 and G3BP1 results in a significant enhancement of TRIM25's ubiquitination activity towards multiple antiviral proteins, which are mainly located in SGs. This co-condensation is critical in activating the RIG-I signaling pathway, thus restraining RNA virus infection. Our studies provide a conceptual framework for better understanding the heterogeneity of stress granule components and their response to distinct environmental stressors.
Subject(s)
RNA Virus Infections , Stress Granules , Tripartite Motif Proteins , Ubiquitin-Protein Ligases , Humans , Cytoplasmic Granules/metabolism , DEAD Box Protein 58/metabolism , DNA Helicases/metabolism , HEK293 Cells , HeLa Cells , Immunity, Innate , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , Receptors, Immunologic/metabolism , RNA Helicases/metabolism , RNA Recognition Motif Proteins/metabolism , RNA Recognition Motif Proteins/genetics , RNA Virus Infections/virology , RNA Virus Infections/metabolism , RNA Virus Infections/immunology , RNA, Double-Stranded/metabolism , Signal Transduction , Stress Granules/metabolism , Transcription Factors/metabolism , Transcription Factors/genetics , Tripartite Motif Proteins/metabolism , Tripartite Motif Proteins/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , UbiquitinationABSTRACT
Acute stress caused by short-term exposure to deleterious chemicals can induce the aggregation of RNA-binding proteins (RBPs) in the cytosol and the formation of stress granules (SGs). The cytoplasmic RBP, Ras GTPase-activating protein-binding protein 1 (G3BP1) is a critical organizer of SG, and its aggregation is considered a hallmark of cellular stress. However, assembly of SG is a highly dynamic process that involves RBPs; hence, existing methods based on fixation processes or overexpression of RBPs exhibit limited efficacy in detecting the assembly of SG under stress conditions. In this study, we established a G3BP1- Green fluorescent protein (GFP) reporter protein in a human neuroblastoma cell line to overcome these limitations. GFP was introduced into the G3BP1 genomic sequence via homologous recombination to generate a G3BP1-GFP fusion protein and further analyze the aggregation processes. We validated the assembly of SG under stress conditions using the G3BP1-GFP reporter system. Additionally, this system supported the evaluation of bisphenol A-induced SG response in the established human neuroblastoma cell line. In conclusion, the established G3BP1-GFP reporter system enables us to monitor the assembly of the SG complex in a human neuroblastoma cell line in real time and can serve as an efficient tool for assessing potential neurotoxicity associated with short-term exposure to chemicals.
Subject(s)
DNA Helicases , Green Fluorescent Proteins , Neuroblastoma , Poly-ADP-Ribose Binding Proteins , RNA Helicases , RNA Recognition Motif Proteins , Humans , RNA Recognition Motif Proteins/genetics , RNA Recognition Motif Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , Poly-ADP-Ribose Binding Proteins/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Cell Line, Tumor , RNA Helicases/genetics , RNA Helicases/metabolism , Neuroblastoma/pathology , DNA Helicases/metabolism , Stress Granules , Stress, Physiological , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/geneticsABSTRACT
Cyclic GMP-AMP synthase (cGAS) undergoes liquid-liquid phase separation (LLPS) to trigger downstream signaling upon double-stranded DNA (dsDNA) stimulation, and the condensed cGAS colocalizes with stress granules (SGs). However, the molecular mechanism underlying the modulation of cGAS activation by SGs remains elusive. In this study, we show that USP8 is localized to SGs upon dsDNA stimulation and potentiates cGAS-stimulator of interferon genes (STING) signaling. A USP8 inhibitor ameliorates pathological inflammation in Trex1-/- mice. Systemic lupus erythematosus (SLE) databases indicate a positive correlation between USP8 expression and SLE. Mechanistic study shows that the SG protein DDX3X promotes cGAS phase separation and activation in a manner dependent on its intrinsic LLPS. USP8 cleaves K27-linked ubiquitin chains from the intrinsically disordered region (IDR) of DDX3X to enhance its condensation. In conclusion, we demonstrate that USP8 catalyzes the deubiquitination of DDX3X to facilitate cGAS condensation and activation and that inhibiting USP8 is a promising strategy for alleviating cGAS-mediated autoimmune diseases.
Subject(s)
DEAD-box RNA Helicases , Interferon Type I , Nucleotidyltransferases , Stress Granules , Ubiquitin Thiolesterase , Ubiquitination , Humans , Animals , Nucleotidyltransferases/metabolism , Ubiquitin Thiolesterase/metabolism , Mice , DEAD-box RNA Helicases/metabolism , Interferon Type I/metabolism , Stress Granules/metabolism , Lupus Erythematosus, Systemic/metabolism , Lupus Erythematosus, Systemic/pathology , Signal Transduction , Mice, Inbred C57BL , HEK293 Cells , Membrane Proteins/metabolism , Mice, Knockout , Exodeoxyribonucleases/metabolism , Endopeptidases , Phosphoproteins , Endosomal Sorting Complexes Required for TransportABSTRACT
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.
Subject(s)
RNA Helicases , RNA Recognition Motif Proteins , RNA, Viral , Stress Granules , Humans , Animals , RNA Recognition Motif Proteins/metabolism , RNA Helicases/metabolism , RNA, Viral/metabolism , Stress Granules/metabolism , SARS-CoV-2/physiology , Immunity, Innate , Signal Transduction , Biomolecular Condensates/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , Virus Diseases/drug therapy , Virus Diseases/metabolism , DNA Helicases/metabolism , eIF-2 Kinase/metabolism , Endoribonucleases/metabolism , COVID-19/virology , COVID-19/immunologyABSTRACT
Upon endoplasmic reticulum (ER) stress, activation of the ER-resident transmembrane protein kinase/endoribonuclease inositol-requiring enzyme 1 (IRE1) initiates a key branch of the unfolded protein response (UPR) through unconventional splicing generation of the transcription factor X-box-binding protein 1 (XBP1s). Activated IRE1 can form large clusters/foci, whose exact dynamic architectures and functional properties remain largely elusive. Here we report that, in mammalian cells, formation of IRE1α clusters is an ER membrane-bound phase separation event that is coupled to the assembly of stress granules (SGs). In response to different stressors, IRE1α clusters are dynamically tethered to SGs at the ER. The cytosolic linker portion of IRE1α possesses intrinsically disordered regions and is essential for its condensation with SGs. Furthermore, disruption of SG assembly abolishes IRE1α clustering and compromises XBP1 mRNA splicing, and such IRE1α-SG coalescence engenders enrichment of the biochemical components of the pro-survival IRE1α-XBP1 pathway during ER stress. Our findings unravel a phase transition mechanism for the spatiotemporal assembly of IRE1α-SG condensates to establish a more efficient IRE1α machinery, thus enabling higher stress-handling capacity.
Subject(s)
Endoplasmic Reticulum Stress , Endoribonucleases , Protein Serine-Threonine Kinases , X-Box Binding Protein 1 , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Endoribonucleases/metabolism , Endoribonucleases/genetics , Humans , X-Box Binding Protein 1/metabolism , X-Box Binding Protein 1/genetics , Animals , RNA Splicing , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/genetics , Stress Granules/metabolism , Stress Granules/genetics , Regulatory Factor X Transcription Factors/metabolism , Regulatory Factor X Transcription Factors/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Unfolded Protein Response , Mice , HeLa Cells , Cytoplasmic Granules/metabolism , Cytoplasmic Granules/genetics , Signal TransductionABSTRACT
Glioblastoma (GBM) is an extremely aggressive primary brain tumor with poor prognosis, short survival time post-diagnosis and high recurrence. Currently, no cure for GBM exists. The identification of an effective therapeutic modality for GBM remains a high priority amongst medical professionals and researches. In recent studies, inhalant cannabidiol (CBD) has demonstrated promise in effectively inhibiting GBM tumor growth. However, exactly how CBD treatment affects the physiology of these tumor cells remains unclear. Stress granules (SG) (a sub-class of biomolecular condensates (BMC)) are dynamic, membrane-less intracellular microstructures which contain proteins and nucleic acids. The formation and signaling of SGs and BMCs plays a significant role in regulating malignancies. This study investigates whether inhaled CBD may play an intervening role towards SGs in GBM tumor cells. Integrated bioinformatics approaches were preformed to gain further insights. This includes use of Immunohistochemistry and flow cytometry to measure SGs, as well as expression and phosphorylation of eukaryotic initiation factor-2α (eIF2α). The findings of this study reveal that CBD receptors (and co-regulated genes) have the potential to play an important biological role in the formation of BMCs within GBM. In this experiment, CBD treatment significantly increased the volume of TIAR-1. This increase directly correlated with elevation in both eIF2α expression and p-eIF2α in CBD treated tissues in comparison to the placebo group (p < 0.05). These results suggest that inhalant CBD significantly up-regulated SGs in GBM, and thus support a theory of targeting BMCs as a potential therapeutic substrate for treating GBM.
Subject(s)
Brain Neoplasms , Cannabidiol , Glioblastoma , Glioblastoma/drug therapy , Glioblastoma/metabolism , Glioblastoma/pathology , Cannabidiol/pharmacology , Humans , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Stress Granules/metabolism , Stress Granules/drug effects , Cell Line, Tumor , Eukaryotic Initiation Factor-2/metabolismABSTRACT
Background: It remains unclear whether BPIV3 infection leads to stress granules formation and whether G3BP1 plays a role in this process and in viral replication. This study aims to clarify the association between BPIV3 and stress granules, explore the effect of G3BP1 on BPIV3 replication, and provide significant insights into the mechanisms by which BPIV3 evades the host's antiviral immunity to support its own survival. Methods: Here, we use Immunofluorescence staining to observe the effect of BPIV3 infection on the assembly of stress granules. Meanwhile, the expression changes of eIF2α and G3BP1 were determined. Overexpression or siRNA silencing of intracellular G3BP1 levels was examined for its regulatory control of BPIV3 replication. Results: We identify that the BPIV3 infection elicited phosphorylation of the eIF2α protein. However, it did not induce the assembly of stress granules; rather, it inhibited the formation of stress granules and downregulated the expression of G3BP1. G3BP1 overexpression facilitated the formation of stress granules within cells and hindered viral replication, while G3BP1 knockdown enhanced BPIV3 expression. Conclusion: This study suggest that G3BP1 plays a crucial role in BPIV3 suppressing stress granule formation and viral replication.
Subject(s)
DNA Helicases , Poly-ADP-Ribose Binding Proteins , RNA Helicases , RNA Recognition Motif Proteins , Stress Granules , Virus Replication , Animals , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , RNA Recognition Motif Proteins/metabolism , DNA Helicases/metabolism , DNA Helicases/genetics , RNA Helicases/metabolism , RNA Helicases/genetics , Stress Granules/metabolism , Cattle , Eukaryotic Initiation Factor-2/metabolism , Respirovirus Infections/immunology , Respirovirus Infections/metabolism , Host-Pathogen Interactions/immunology , Phosphorylation , Cell Line , Cytoplasmic Granules/metabolismABSTRACT
Stress granules (SGs) are dynamic membraneless organelles that form in response to cellular stress. SGs are predominantly composed of RNA and RNA-binding proteins that assemble through liquid-liquid phase separation. Although the formation of SGs is considered a transient and protective response to cellular stress, their dysregulation or persistence may contribute to various neurodegenerative diseases. This review aims to provide a comprehensive overview of SG physiology and pathology. It covers the formation, composition, regulation, and functions of SGs, along with their crosstalk with other membrane-bound and membraneless organelles. Furthermore, this review discusses the dual roles of SGs as both friends and foes in neurodegenerative diseases and explores potential therapeutic approaches targeting SGs. The challenges and future perspectives in this field are also highlighted. A more profound comprehension of the intricate relationship between SGs and neurodegenerative diseases could inspire the development of innovative therapeutic interventions against these devastating diseases.
Subject(s)
Neurodegenerative Diseases , Stress Granules , Humans , Neurodegenerative Diseases/metabolism , Animals , Stress Granules/metabolism , Cytoplasmic Granules/metabolismABSTRACT
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.
Subject(s)
Antineoplastic Agents , Sorafenib , Stress Granules , Humans , Sorafenib/pharmacology , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Stress Granules/metabolism , HeLa Cells , Drug Resistance, Neoplasm/drug effects , Peptides/pharmacology , Peptides/chemistry , Cell Survival/drug effects , Ubiquitin Thiolesterase/metabolism , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Cell Line, Tumor , Cell-Penetrating Peptides/pharmacology , Cell-Penetrating Peptides/chemistryABSTRACT
Zonula occludens-1 (ZO-1) is involved in the regulation of cell-cell junctions between endothelial cells (ECs). Here we identify the ZO-1 protein interactome and uncover ZO-1 interactions with RNA-binding proteins that are part of stress granules (SGs). Downregulation of ZO-1 increased SG formation in response to stress and protected ECs from cellular insults. The ZO-1 interactome uncovered an association between ZO-1 and Y-box binding protein 1 (YB-1), a constituent of SGs. Arsenite treatment of ECs decreased the interaction between ZO-1 and YB-1, and drove SG assembly. YB-1 expression is essential for SG formation and for the cytoprotective effects induced by ZO-1 downregulation. In the developing retinal vascular plexus of newborn mice, ECs at the front of growing vessels express less ZO-1 but display more YB-1-positive granules than ECs located in the vascular plexus. Endothelial-specific deletion of ZO-1 in mice at post-natal day 7 markedly increased the presence of YB-1-positive granules in ECs of retinal blood vessels, altered tip EC morphology and vascular patterning, resulting in aberrant endothelial proliferation, and arrest in the expansion of the retinal vasculature. Our findings suggest that, through its interaction with YB-1, ZO-1 controls SG formation and the response of ECs to stress during angiogenesis.