RESUMO
Complex diseases often involve the interplay between genetic and environmental factors. Charcot-Marie-Tooth type 2 neuropathies (CMT2) are a group of genetically heterogeneous disorders, in which similar peripheral neuropathology is inexplicably caused by various mutated genes. Their possible molecular links remain elusive. Here, we found that upon environmental stress, many CMT2-causing mutant proteins adopt similar properties by entering stress granules (SGs), where they aberrantly interact with G3BP and integrate into SG pathways. For example, glycyl-tRNA synthetase (GlyRS) is translocated from the cytoplasm into SGs upon stress, where the mutant GlyRS perturbs the G3BP-centric SG network by aberrantly binding to G3BP. This disrupts SG-mediated stress responses, leading to increased stress vulnerability in motoneurons. Disrupting this aberrant interaction rescues SG abnormalities and alleviates motor deficits in CMT2D mice. These findings reveal a stress-dependent molecular link across diverse CMT2 mutants and provide a conceptual framework for understanding genetic heterogeneity in light of environmental stress.
Assuntos
Doença de Charcot-Marie-Tooth , Proteínas com Motivo de Reconhecimento de RNA , Grânulos de Estresse , Animais , Camundongos , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Citoplasma , Neurônios Motores , Proteínas com Motivo de Reconhecimento de RNA/metabolismoRESUMO
N7-methylguanosine (m7G) modification, routinely occurring at mRNA 5' cap or within tRNAs/rRNAs, also exists internally in messenger RNAs (mRNAs). Although m7G-cap is essential for pre-mRNA processing and protein synthesis, the exact role of mRNA internal m7G modification remains elusive. Here, we report that mRNA internal m7G is selectively recognized by Quaking proteins (QKIs). By transcriptome-wide profiling/mapping of internal m7G methylome and QKI-binding sites, we identified more than 1,000 high-confidence m7G-modified and QKI-bound mRNA targets with a conserved "GANGAN (N = A/C/U/G)" motif. Strikingly, QKI7 interacts (via C terminus) with the stress granule (SG) core protein G3BP1 and shuttles internal m7G-modified transcripts into SGs to regulate mRNA stability and translation under stress conditions. Specifically, QKI7 attenuates the translation efficiency of essential genes in Hippo signaling pathways to sensitize cancer cells to chemotherapy. Collectively, we characterized QKIs as mRNA internal m7G-binding proteins that modulate target mRNA metabolism and cellular drug resistance.
Assuntos
DNA Helicases , RNA Helicases , DNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA Helicases/metabolismo , Grânulos de Estresse , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação ao GTP/metabolismo , RNA Mensageiro/metabolismo , Grânulos Citoplasmáticos/metabolismoRESUMO
Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , DNA Helicases/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Esclerose Tuberosa/metabolismo , Sequência de Aminoácidos , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/química , Evolução Molecular , Feminino , Humanos , Insulina/farmacologia , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenótipo , Proteínas de Ligação a Poli-ADP-Ribose/química , RNA Helicases/química , Proteínas com Motivo de Reconhecimento de RNA/química , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra/metabolismoRESUMO
Stressed cells shut down translation, release mRNA molecules from polysomes, and form stress granules (SGs) via a network of interactions that involve G3BP. Here we focus on the mechanistic underpinnings of SG assembly. We show that, under non-stress conditions, G3BP adopts a compact auto-inhibited state stabilized by electrostatic intramolecular interactions between the intrinsically disordered acidic tracts and the positively charged arginine-rich region. Upon release from polysomes, unfolded mRNAs outcompete G3BP auto-inhibitory interactions, engendering a conformational transition that facilitates clustering of G3BP through protein-RNA interactions. Subsequent physical crosslinking of G3BP clusters drives RNA molecules into networked RNA/protein condensates. We show that G3BP condensates impede RNA entanglement and recruit additional client proteins that promote SG maturation or induce a liquid-to-solid transition that may underlie disease. We propose that condensation coupled to conformational rearrangements and heterotypic multivalent interactions may be a general principle underlying RNP granule assembly.
Assuntos
Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Células HeLa , Humanos , Conformação de Ácido Nucleico , Organelas/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Estresse Fisiológico/genéticaRESUMO
Liquid-liquid phase separation (LLPS) mediates formation of membraneless condensates such as those associated with RNA processing, but the rules that dictate their assembly, substructure, and coexistence with other liquid-like compartments remain elusive. Here, we address the biophysical mechanism of this multiphase organization using quantitative reconstitution of cytoplasmic stress granules (SGs) with attached P-bodies in human cells. Protein-interaction networks can be viewed as interconnected complexes (nodes) of RNA-binding domains (RBDs), whose integrated RNA-binding capacity determines whether LLPS occurs upon RNA influx. Surprisingly, both RBD-RNA specificity and disordered segments of key proteins are non-essential, but modulate multiphase condensation. Instead, stoichiometry-dependent competition between protein networks for connecting nodes determines SG and P-body composition and miscibility, while competitive binding of unconnected proteins disengages networks and prevents LLPS. Inspired by patchy colloid theory, we propose a general framework by which competing networks give rise to compositionally specific and tunable condensates, while relative linkage between nodes underlies multiphase organization.
Assuntos
Grânulos Citoplasmáticos/fisiologia , Estruturas Citoplasmáticas/fisiologia , Mapas de Interação de Proteínas/fisiologia , Fenômenos Biofísicos , Linhagem Celular Tumoral , Citoplasma/metabolismo , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Extração Líquido-Líquido/métodos , Organelas/química , RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/fisiologiaRESUMO
The mechanisms underlying ribonucleoprotein (RNP) granule assembly, including the basis for establishing and maintaining RNP granules with distinct composition, are unknown. One prominent type of RNP granule is the stress granule (SG), a dynamic and reversible cytoplasmic assembly formed in eukaryotic cells in response to stress. Here, we show that SGs assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein-RNA interaction network. The central node of this network is G3BP1, which functions as a molecular switch that triggers RNA-dependent LLPS in response to a rise in intracellular free RNA concentrations. Moreover, we show that interplay between three distinct intrinsically disordered regions (IDRs) in G3BP1 regulates its intrinsic propensity for LLPS, and this is fine-tuned by phosphorylation within the IDRs. Further regulation of SG assembly arises through positive or negative cooperativity by extrinsic G3BP1-binding factors that strengthen or weaken, respectively, the core SG network.
Assuntos
Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Ribonucleoproteínas/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Estruturas Citoplasmáticas/metabolismo , Células HEK293 , Humanos , Fosforilação , RNA/metabolismoRESUMO
Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.
Assuntos
Grânulos Citoplasmáticos/genética , DNA Helicases/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Biossíntese de Proteínas , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Estresse Fisiológico/genética , Regiões 5' não Traduzidas , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/patologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Feminino , Células HCT116 , Células HeLa , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Espermatogônias/citologia , Espermatogônias/patologia , Testículo/citologia , Testículo/metabolismoRESUMO
Post-transcriptional mechanisms regulate the stability and, hence, expression of coding and noncoding RNAs. Sequence-specific features within the 3' untranslated region (3' UTR) often direct mRNAs for decay. Here, we characterize a genome-wide RNA decay pathway that reduces the half-lives of mRNAs based on overall 3' UTR structure formed by base pairing. The decay pathway is independent of specific single-stranded sequences, as regulation is maintained in both the original and reverse complement orientation. Regulation can be compromised by reducing the overall structure by fusing the 3' UTR with an unstructured sequence. Mutating base-paired RNA regions can also compromise this structure-mediated regulation, which can be restored by re-introducing base-paired structures of different sequences. The decay pathway requires the RNA-binding protein UPF1 and its associated protein G3BP1. Depletion of either protein increased steady-state levels of mRNAs with highly structured 3' UTRs as well as highly structured circular RNAs. This structure-dependent mechanism therefore enables cells to selectively regulate coding and noncoding RNAs.
Assuntos
Regiões 3' não Traduzidas , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Transativadores/metabolismo , Pareamento de Bases , Linhagem Celular , Regulação da Expressão Gênica , Humanos , RNA Circular/química , RNA Circular/metabolismoRESUMO
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.
Assuntos
Corpos de Processamento , Grânulos de Estresse , Grânulos Citoplasmáticos , RNA Helicases , CitoplasmaRESUMO
Filamin A (FLNA) is an actin crosslinking protein that mediates mechanotransduction. External and internal mechanical forces, through the actin cytoskeleton, can induce conformational changes of the FLNA molecule to expose cryptic binding sites for its binding partners. Here, we identified Ras GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) as a new FLNA mechanobinding partner. Unlike other FLNA binding partners to the mechanosensing domain repeat 21 (R21), G3BP1 requires an additional neighboring repeat R22 to interact. We demonstrated that their interaction occurs in the cytosol of living cells in an actin polymerization-dependent manner. We also mapped the FLNA-binding site on G3BP1 and found that a F360A point mutation in the RNA recognition motif disrupts the interaction. RNA interfered with the FLNA-G3BP1 interaction, and FLNA did not localize in RNA-rich stress granules (SGs). Disruption of the interaction was sufficient to promote phase-separated SG formation, and arsenite treatment further stimulated the formation of SGs. Taken together, these data identify G3BP1 as a new mechanobinding protein that interacts with the FLNA mechanosensing domain R21 and suggest that SG formation is partially regulated by mechanical force.
Assuntos
Actinas , DNA Helicases , Filaminas/metabolismo , Actinas/metabolismo , Grânulos de Estresse , Mecanotransdução Celular , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNARESUMO
SIRT2, a cytoplasmic member of the Sirtuin family, has important roles in immunity and inflammation. However, its function in regulating the response to DNA virus infection remains elusive. Here, we find that SIRT2 is a unique regulator among the Sirtuin family that negatively modulates the cGAS-STING-signaling pathway. SIRT2 is down-regulated after Herpes simplex virus-1 (HSV-1) infection, and SIRT2 deficiency markedly elevates the expression levels of type I interferon (IFN). SIRT2 inhibits the DNA binding ability and droplet formation of cGAS by interacting with and deacetylating G3BP1 at K257, K276, and K376, leading to the disassembly of the cGAS-G3BP1 complex, which is critical for cGAS activation. Administration of AGK2, a selective SIRT2 inhibitor, protects mice from HSV-1 infection and increases the expression of IFN and IFN-stimulated genes. Our study shows that SIRT2 negatively regulates cGAS activation through G3BP1 deacetylation, suggesting a potential antiviral strategy by modulating SIRT2 activity.
Assuntos
DNA Helicases , Imunidade Inata , Animais , Camundongos , DNA Helicases/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Transdução de Sinais , Sirtuína 2/genética , Sirtuína 2/metabolismoRESUMO
Stress granules (SGs) are cytoplasmic biomolecular condensates containing proteins and RNAs in response to stress. Ras-GTPase-activating protein binding protein 1 (G3BP1) is a core SG protein. Caprin-1 and ubiquitin specific peptidase 10 (USP10) interact with G3BP1, facilitating and suppressing SG formation, respectively. The crystal structures of the nuclear transport factor 2-like (NTF2L) domain of G3BP1 in complex with the G3BP1-interacting motif (GIM) of Caprin-1 and USP10 show that both GIMs bind to the same hydrophobic pocket of G3BP1. Moreover, both GIMs suppressed the liquid-liquid phase separation (LLPS) of G3BP1, suggesting that Caprin-1 likely facilitates SG formation via other mechanisms. Thus, we dissected various domains of Caprin-1 and investigated their role in LLPS in vitro and SG formation in cells. The C-terminal domain of Caprin-1 underwent spontaneous LLPS, whereas the N-terminal domain and GIM of Caprin-1 suppressed LLPS of G3BP1. The opposing effect of the N- and C-terminal domains of Caprin-1 on SG formation were demonstrated in cells with or without the endogenous Caprin-1. We propose that the N- and C-terminal domains of Caprin-1 regulate SG formation in a "yin and yang" fashion, mediating the dynamic and reversible assembly of SGs.
Assuntos
DNA Helicases , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , DNA Helicases/metabolismo , Grânulos Citoplasmáticos/metabolismo , Grânulos de Estresse , Proteínas Ativadoras de GTPase/metabolismo , Proteases Específicas de Ubiquitina/metabolismoRESUMO
Stress granules are the RNA/protein condensates assembled in the cells under stress. They play a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, how stress granule assembly is regulated and related to ALS/FTD pathomechanism is incompletely understood. Mutation in the C9orf72 gene is the most common cause of familial ALS and FTD. C9orf72 mutation causes the formation of toxic dipeptide repeats. Here we show that the two most toxic dipeptide repeats [i.e., poly(GR) and poly(PR)] activate c-Jun N-terminal kinase (JNK) via the ER-stress response protein IRE1 using fly and cellular models. Further, we show that activated JNK promotes stress granule assembly in cells by promoting the transcription of one of the key stress granule proteins (i.e., G3BP1) by inducing histone 3 phosphorylation. Consistent with these findings, JNK or IRE1 inhibition reduced stress granule formation, histone 3 phosphorylation, G3BP1 mRNA and protein levels, and neurotoxicity in cells overexpressing poly(GR) and poly(PR) or neurons derived from male and female C9ALS/FTD patient-induced pluripotent stem cells. Our findings connect ER stress, JNK activation, and stress granule assembly in a unified pathway contributing to C9ALS/FTD neurodegeneration.SIGNIFICANCE STATEMENT c-Jun N-terminal kinase (JNK) is a part of the mitogen-activated protein kinase pathway, which is the central node for the integration of multiple stress signals. Cells are under constant stress in neurodegenerative diseases, and how these cells respond to stress signals is a critical factor in determining their survival or death. Previous studies have shown JNK as a major contributor to cellular apoptosis. Here, we show the role of JNK in stress granule assembly. We identify that toxic dipeptide repeats produced in ALS/FTD conditions activate JNK. The activated JNK in the nucleus can induce histone modifications which increase G3BP1 expression, thus promoting stress granule assembly and neurodegeneration.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Feminino , Humanos , Masculino , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Proteína C9orf72/genética , Dipeptídeos/genética , Dipeptídeos/metabolismo , DNA Helicases/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Histonas , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA , Grânulos de Estresse , Drosophila melanogaster , AnimaisRESUMO
Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs are regulated by different stimulators including viral infection, which is involved in the antiviral activity of host cells to limit viral propagation. To survive, several viruses have been reported to execute various strategies, such as modulating SG formation, to create optimal surroundings for viral replication. African swine fever virus (ASFV) is one of the most notorious pathogens in the global pig industry. However, the interplay between ASFV infection and SG formation remains largely unknown. In this study, we found that ASFV infection inhibited SG formation. Through SG inhibitory screening, we found that several ASFV-encoded proteins are involved in inhibition of SG formation. Among them, an ASFV S273R protein (pS273R), the only cysteine protease encoded by the ASFV genome, significantly affected SG formation. ASFV pS273R interacted with G3BP1 (Ras-GTPase-activating protein [SH3 domain] binding protein 1), a vital nucleating protein of SG formation. Furthermore, we found that ASFV pS273R cleaved G3BP1 at the G140-F141 to produce two fragments (G3BP1-N1-140 and G3BP1-C141-456). Interestingly, both the pS273R-cleaved fragments of G3BP1 lost the ability to induce SG formation and antiviral activity. Taken together, our finding reveals that the proteolytic cleavage of G3BP1 by ASFV pS273R is a novel mechanism by which ASFV counteracts host stress and innate antiviral responses.
Assuntos
Vírus da Febre Suína Africana , Grânulos de Estresse , Proteínas Virais , Animais , Febre Suína Africana/metabolismo , Febre Suína Africana/virologia , Vírus da Febre Suína Africana/enzimologia , Vírus da Febre Suína Africana/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Grânulos de Estresse/metabolismo , Suínos , Replicação Viral/fisiologia , Chlorocebus aethiops , Humanos , Células HEK293 , Células Cultivadas , Macrófagos Alveolares/virologia , Proteínas Virais/metabolismo , ProteóliseRESUMO
Aberrant expression of forkhead box transcription factor 1 (FOXM1) plays critical roles in a variety of human malignancies and predicts poor prognosis. However, little is known about the crosstalk between FOXM1 and long noncoding RNAs (lncRNAs) in tumorigenesis. The present study identifies a previously uncharacterized lncRNA XLOC_008672 in gastric cancer (GC), which is regulated by FOXM1 and possesses multiple copies of tandem repetitive sequences. LncRNA microarrays are used to screen differentially expressed lncRNAs in FOXM1 knockdown GC cells, and then the highest fold downregulation lncRNA XLOC_008672 is screened out. Sequence analysis reveals that the new lncRNA contains 62 copies of 37-bp tandem repeats. It is transcriptionally activated by FOXM1 and functions as a downstream effector of FOXM1 in GC cells through in vitro and in vivo functional assays. Elevated expression of XLOC_008672 is found in GC tissues and indicates worse prognosis. Mechanistically, XLOC_008672 can bind to small nuclear ribonucleoprotein polypeptide A (SNRPA), thereby enhancing mRNA stability of Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) and, consequently, facilitating GC cell proliferation and migration. Our study discovers a new uncharacterized lncRNA XLOC_008672 involved in GC carcinogenesis and progression. Targeting FOXM1/XLOC_008672/SNRPA/G3BP1 signaling axis might be a promising therapeutic strategy for GC.
Assuntos
Carcinogênese , Proliferação de Células , Proteína Forkhead Box M1 , Regulação Neoplásica da Expressão Gênica , RNA Longo não Codificante , Neoplasias Gástricas , Animais , Feminino , Humanos , Masculino , Camundongos , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , DNA Helicases , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Camundongos Nus , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Prognóstico , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Neoplasias Gástricas/metabolismo , Sequências de Repetição em Tandem/genéticaRESUMO
Mitochondria house anabolic and catabolic processes that must be balanced and adjusted to meet cellular demands. The RNA-binding protein CLUH (clustered mitochondria homolog) binds mRNAs of nuclear-encoded mitochondrial proteins and is highly expressed in the liver, where it regulates metabolic plasticity. Here, we show that in primary hepatocytes, CLUH coalesces in specific ribonucleoprotein particles that define the translational fate of target mRNAs, such as Pcx, Hadha, and Hmgcs2, to match nutrient availability. Moreover, CLUH granules play signaling roles, by recruiting mTOR kinase and the RNA-binding proteins G3BP1 and G3BP2. Upon starvation, CLUH regulates translation of Hmgcs2, involved in ketogenesis, inhibits mTORC1 activation and mitochondrial anabolic pathways, and promotes mitochondrial turnover, thus allowing efficient reprograming of metabolic function. In the absence of CLUH, a mitophagy block causes mitochondrial clustering that is rescued by rapamycin treatment or depletion of G3BP1 and G3BP2. Our data demonstrate that metabolic adaptation of liver mitochondria to nutrient availability depends on a compartmentalized CLUH-dependent post-transcriptional mechanism that controls both mTORC1 and G3BP signaling and ensures survival.
Assuntos
Mitocôndrias Hepáticas/fisiologia , Proteínas Mitocondriais/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Animais , Células COS , Chlorocebus aethiops , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Mitofagia , Proteínas de Ligação a RNA/genéticaRESUMO
The herpes simplex virus 1 (HSV1) virion host shutoff (vhs) protein is an endoribonuclease that regulates the translational environment of the infected cell, by inducing the degradation of host mRNA via cellular exonuclease activity. To further understand the relationship between translational shutoff and mRNA decay, we have used ectopic expression to compare HSV1 vhs (vhsH) to its homologues from four other alphaherpesviruses - varicella zoster virus (vhsV), bovine herpesvirus 1 (vhsB), equine herpesvirus 1 (vhsE) and Marek's disease virus (vhsM). Only vhsH, vhsB and vhsE induced degradation of a reporter luciferase mRNA, with poly(A)+ in situ hybridization indicating a global depletion of cytoplasmic poly(A)+ RNA and a concomitant increase in nuclear poly(A)+ RNA and the polyA tail binding protein PABPC1 in cells expressing these variants. By contrast, vhsV and vhsM failed to induce reporter mRNA decay and poly(A)+ depletion, but rather, induced cytoplasmic G3BP1 and poly(A)+ mRNA- containing granules and phosphorylation of the stress response proteins eIF2α and protein kinase R. Intriguingly, regardless of their apparent endoribonuclease activity, all vhs homologues induced an equivalent general blockade to translation as measured by single-cell puromycin incorporation. Taken together, these data suggest that the activities of translational arrest and mRNA decay induced by vhs are separable and we propose that they represent sequential steps of the vhs host interaction pathway.
Assuntos
Herpesvirus Humano 1 , Proteínas Virais , Proteínas Virais/genética , Proteínas Virais/metabolismo , Ribonucleases , DNA Helicases , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Herpesvirus Humano 1/genética , Endorribonucleases/metabolismo , Estabilidade de RNA , Vírion/genética , Vírion/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
To rapidly adapt to stresses such as infections, cells have evolved several mechanisms, which include the activation of stress response pathways and the innate immune response. These stress responses result in the rapid inhibition of translation and condensation of stalled mRNAs with RNA-binding proteins and signalling components into cytoplasmic biocondensates called stress granules (SGs). Increasing evidence suggests that SGs contribute to antiviral defence, and thus viruses need to evade these responses to propagate. We previously showed that feline calicivirus (FCV) impairs SG assembly by cleaving the scaffolding protein G3BP1. We also observed that uninfected bystander cells assembled G3BP1-positive granules, suggesting a paracrine response triggered by infection. We now present evidence that virus-free supernatant generated from infected cells can induce the formation of SG-like foci, which we name paracrine granules. They are linked to antiviral activity and exhibit specific kinetics of assembly-disassembly, and protein and RNA composition that are different from canonical SGs. We propose that this paracrine induction reflects a novel cellular defence mechanism to limit viral propagation and promote stress responses in bystander cells.
Assuntos
Infecções por Caliciviridae , Grânulos de Estresse , Animais , Infecções por Caliciviridae/imunologia , Calicivirus Felino/imunologia , Gatos , Proteínas de Ligação a Poli-ADP-Ribose/imunologia , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Grânulos de Estresse/imunologia , Replicação Viral/fisiologiaRESUMO
Stress granule (SG) is a temporary cellular structure that plays a crucial role in the regulation of mRNA and protein sequestration during various cellular stress conditions. SG enables cells to cope with stress more effectively, conserving vital energy and resources. Focusing on the NTF2-like domain of G3BP1, a key protein in SG dynamics, we explore to identify and characterize novel small molecules involved in SG modulation without external stressors. Through in silico molecular docking approach to simulate the interaction between various compounds and the NTF2-like domain of G3BP1, we identified three compounds as potential candidates that could bind to the NTF2-like domain of G3BP1. Subsequent immunofluorescence experiments demonstrated that these compounds induce the formation of SG-like, G3BP1-positive granules. Importantly, the granule formation by these compounds occurs independent from the phosphorylation of eIF2α, a common mechanism in SG formation, suggesting that it might offer a new strategy for influencing SG dynamics implicated in various diseases.
Assuntos
DNA Helicases , RNA Helicases , DNA Helicases/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Simulação de Acoplamento Molecular , Grânulos Citoplasmáticos/metabolismoRESUMO
IMPORTANCE: Our study highlights the mechanisms behind the cell's resistance to stress granule (SG) formation after infection with Old World alphaviruses. Shortly after infection, the replication of these viruses hinders the cell's ability to form SGs, even when exposed to chemical inducers such as sodium arsenite. This resistance is primarily attributed to virus-induced transcriptional and translational shutoffs, rather than interactions between the viral nsP3 and the key components of SGs, G3BP1/2, or the ADP-ribosylhydrolase activity of nsP3 macro domain. While interactions between G3BPs and nsP3 are essential for the formation of viral replication complexes, their role in regulating SG development appears to be small, if any. Cells harboring replicating viruses or replicons with lower abilities to inhibit transcription and/or translation, but expressing wild-type nsP3, retain the ability for SG development. Understanding these mechanisms of regulation of SG formation contributes to our knowledge of viral replication and the intricate relationships between alphaviruses and host cells.