RESUMEN
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.
Asunto(s)
ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Gránulos de Estrés , Humanos , Gránulos de Estrés/metabolismo , Gránulos de Estrés/genética , ARN Helicasas/metabolismo , ARN Helicasas/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Cuerpos de Procesamiento/metabolismo , Cuerpos de Procesamiento/genética , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Gránulos Citoplasmáticos/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Células HeLa , ADN Helicasas/metabolismo , ADN Helicasas/genética , Células HEK293 , Unión Proteica , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Proteínas Proto-OncogénicasRESUMEN
In plants, nucleotide-binding site and leucine-rich repeat proteins (NLRs) play pivotal roles in effector-triggered immunity (ETI). However, the precise mechanisms underlying NLR-mediated disease resistance remain elusive. Previous studies have demonstrated that the NLR gene pair Pik-H4 confers resistance to rice blast disease by interacting with the transcription factor OsBIHD1, consequently leading to the upregulation of hormone pathways. In the present study, we identified an RNA recognition motif (RRM) protein, OsRRM2, which interacted with Pik1-H4 and Pik2-H4 in vesicles and chloroplasts. OsRRM2 exhibited a modest influence on Pik-H4-mediated rice blast resistance by upregulating resistance genes and genes associated with chloroplast immunity. Moreover, the RNA-binding sequence of OsRRM2 was elucidated using systematic evolution of ligands by exponential enrichment. Transcriptome analysis further indicated that OsRRM2 promoted RNA editing of the chloroplastic gene ndhB. Collectively, our findings uncovered a chloroplastic RRM protein that facilitated the translocation of the NLR gene pair and modulated chloroplast immunity, thereby bridging the gap between ETI and chloroplast immunity.
Asunto(s)
Cloroplastos , Regulación de la Expresión Génica de las Plantas , Oryza , Inmunidad de la Planta , Proteínas de Plantas , Cloroplastos/metabolismo , Cloroplastos/genética , Inmunidad de la Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Oryza/genética , Oryza/metabolismo , Oryza/inmunología , Proteínas Repetidas Ricas en Leucina , Sitios de Unión , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Resistencia a la Enfermedad/genética , Proteínas NLR/metabolismo , Proteínas NLR/genética , Edición de ARNRESUMEN
The skin, the organ with the largest surface area in the body, is the most susceptible to chemical exposure from the external environment. In this study, we aimed to establish an in vitro skin toxicity monitoring system that utilizes the mechanism of stress granule (SG) formation induced by various cellular stresses. In HaCaT cells, a keratinocyte cell line that comprises the human skin, a green fluorescent protein (GFP) was knocked in at the C-terminal genomic locus of Ras GTPase-activating protein-binding protein 1 (G3BP1), a representative component of SGs. The G3BP1-GFP knock-in HaCaT cells and wild-type (WT) HaCaT cells formed SGs containing G3BP1-GFP upon exposure to arsenite and household chemicals, such as bisphenol A (BPA) and benzalkonium chloride (BAC), in real-time. In addition, the exposure of G3BP1-GFP knock-in HaCaT cells to BPA and BAC promoted the phosphorylation of eukaryotic initiation factor 2 alpha and protein kinase R-like endoplasmic reticulum kinase, which are cell signaling factors involved in SG formation, similar to WT HaCaT cells. In conclusion, this novel G3BP1-GFP knock-in human skin cell system can monitor SG formation in real-time and be utilized to assess skin toxicity to various substances.
Asunto(s)
Gránulos Citoplasmáticos , ADN Helicasas , Proteínas Fluorescentes Verdes , Queratinocitos , Proteínas de Unión a Poli-ADP-Ribosa , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Humanos , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Arsenitos/toxicidad , Piel/efectos de los fármacos , Piel/metabolismo , Técnicas de Sustitución del Gen , Genes Reporteros/efectos de los fármacos , Fenoles/toxicidad , Células HaCaT , Fosforilación , Compuestos de Bencidrilo/toxicidad , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Pruebas de Toxicidad/métodosRESUMEN
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.
Asunto(s)
ADN Helicasas , Proteínas Fluorescentes Verdes , Neuroblastoma , Proteínas de Unión a Poli-ADP-Ribosa , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Humanos , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Línea Celular Tumoral , ARN Helicasas/genética , ARN Helicasas/metabolismo , Neuroblastoma/patología , ADN Helicasas/metabolismo , Gránulos de Estrés , Estrés Fisiológico , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
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.
Asunto(s)
Infecciones por Virus ARN , Gránulos de Estrés , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas , Humanos , Gránulos Citoplasmáticos/metabolismo , Proteína 58 DEAD Box/metabolismo , ADN Helicasas/metabolismo , Células HEK293 , Células HeLa , Inmunidad Innata , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Receptores Inmunológicos/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/genética , Infecciones por Virus ARN/virología , Infecciones por Virus ARN/metabolismo , Infecciones por Virus ARN/inmunología , ARN Bicatenario/metabolismo , Transducción de Señal , Gránulos de Estrés/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas de Motivos Tripartitos/metabolismo , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Stress granules (SGs), the main component is GTPase-activating protein-binding protein 1 (G3BP1), which are assembled during viral infection and function to sequester host and viral mRNAs and proteins, are part of the antiviral responses. In this study, we found that porcine deltacoronavirus (PDCoV) infection induced stable formation of robust SGs in cells through a PERK (protein kinase R-like endoplasmic reticulum kinase)-dependent mechanism. Overexpression of SGs marker proteins G3BP1 significantly reduced PDCoV replication in vitro, while inhibition of endogenous G3BP1 enhanced PDCoV replication. Moreover, PDCoV infected LLC-PK1 cells raise the phosphorylation level of G3BP1. By overexpression of the G3BP1 phosphorylated protein or the G3BP1 dephosphorylated protein, we found that phosphorylation of G3BP1 is involved in the regulation of PDCoV-induced inflammatory response. Taken together, our study presents a vital aspect of the host innate response to invading pathogens and reveals attractive host targets for antiviral target.
Asunto(s)
ADN Helicasas , Inflamación , Proteínas de Unión a Poli-ADP-Ribosa , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Animales , Porcinos , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Fosforilación , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Helicasas/metabolismo , ARN Helicasas/genética , ADN Helicasas/metabolismo , ADN Helicasas/genética , Replicación Viral , Coronavirus/inmunología , Coronavirus/fisiología , Línea Celular , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/genética , Inmunidad InnataRESUMEN
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.
Asunto(s)
Carcinogénesis , Proliferación Celular , Proteína Forkhead Box M1 , Regulación Neoplásica de la Expresión Génica , ARN Largo no Codificante , Neoplasias Gástricas , Animales , Femenino , Humanos , Masculino , Ratones , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , ADN Helicasas , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Ratones Desnudos , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Pronóstico , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Neoplasias Gástricas/metabolismo , Secuencias Repetidas en Tándem/genéticaRESUMEN
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.
Asunto(s)
Neoplasias de los Conductos Biliares , Colangiocarcinoma , Progresión de la Enfermedad , Escherichia coli , FN-kappa B , Gránulos de Estrés , Animales , Humanos , Ratones , Neoplasias de los Conductos Biliares/metabolismo , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/patología , Colangiocarcinoma/metabolismo , Colangiocarcinoma/genética , Colangiocarcinoma/patología , Modelos Animales de Enfermedad , ADN Helicasas , Escherichia coli/genética , Escherichia coli/metabolismo , Gemcitabina , FN-kappa B/metabolismo , FN-kappa B/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/genética , Transducción de Señal/genética , Gránulos de Estrés/metabolismo , Gránulos de Estrés/genéticaRESUMEN
Ras-GTPase-activating protein (GAP)-binding protein 1 (G3BP1) is an RNA-binding protein implicated in various malignancies. However, its role in nasopharyngeal carcinoma (NPC) remains elusive. This study elucidates the potential regulation mechanisms of G3BP1 and its significance in NPC advancement. Through knockdown and overexpression approaches, we validate G3BP1's oncogenic role by promoting proliferation, migration, and invasion in vitro and in vivo. Moreover, G3BP1 emerges as a key regulator of the JAK2/STAT3 signaling pathway, augmenting JAK2 expression via mRNA binding. Notably, epigallocatechin gallate (EGCG), a green tea-derived antioxidant, counteracts G3BP1-mediated pathway activation. Clinical analysis reveals heightened G3BP1, JAK2, and p-STAT3 as powerful prognostic markers, with G3BP1's expression standing as an independent indicator of poorer outcomes for NPC patients. In conclusion, the study unveils the oncogenic prowess of G3BP1, its orchestration of the JAK2/STAT3 signaling pathway, and its pivotal role in NPC progression.
Asunto(s)
ADN Helicasas , Neoplasias Nasofaríngeas , Humanos , Carcinoma Nasofaríngeo/genética , ADN Helicasas/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Línea Celular Tumoral , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Transducción de Señal/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patología , Proliferación Celular/genética , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismoRESUMEN
Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the RNA-binding proteins G3BP1/2. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting the co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress and dissolve pre-existing stress granules. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent powerful tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.
Asunto(s)
ADN Helicasas , ARN Helicasas , Gránulos de Estrés , ADN Helicasas/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Helicasas/genética , Proteínas con Motivos de Reconocimiento de ARN/genéticaRESUMEN
BACKGROUND: Most patients with ovarian cancer (OC) treated with platinum-based chemotherapy have a dismal prognosis owing to drug resistance. However, the regulatory mechanisms of circular RNA (circRNA) and p53 ubiquitination are unknown in platinum-resistant OC. We aimed to identify circRNAs associated with platinum-resistant OC to develop a novel treatment strategy. METHODS: Platinum-resistant circRNAs were screened through circRNA sequencing and validated using quantitative reverse-transcription PCR in OC cells and tissues. The characteristics of circNUP50 were analysed using Sanger sequencing, oligo (dT) primers, ribonuclease R and fluorescence in situ hybridisation assays. Functional experimental studies were performed in vitro and in vivo. The mechanism underlying circNUP50-mediated P53 ubiquitination was investigated through circRNA pull-down analysis and mass spectrometry, luciferase reporters, RNA binding protein immunoprecipitation, immunofluorescence assays, cycloheximide chase assays, and ubiquitination experiments. Finally, a platinum and si-circNUP50 co-delivery nanosystem (Psc@DPP) was constructed to treat platinum-resistant OC in an orthotopic animal model. RESULTS: We found that circNUP50 contributes to platinum-resistant conditions in OC by promoting cell proliferation, affecting the cell cycle, and reducing apoptosis. The si-circNUP50 mRNA sequencing and circRNA pull-down analysis showed that circNUP50 mediates platinum resistance in OC by binding p53 and UBE2T, accelerating p53 ubiquitination. By contrast, miRNA sequencing and circRNA pull-down experiments indicated that circNUP50 could serve as a sponge for miR-197-3p, thereby upregulating G3BP1 to mediate p53 ubiquitination, promoting OC platinum resistance. Psc@DPP effectively overcame platinum resistance in an OC tumour model and provided a novel idea for treating platinum-resistant OC using si-circNUP50. CONCLUSIONS: This study reveals a novel molecular mechanism by which circNUP50 mediates platinum resistance in OC by modulating p53 ubiquitination and provides new insights for developing effective therapeutic strategies for platinum resistance in OC.
Asunto(s)
MicroARNs , Neoplasias Ováricas , Enzimas Ubiquitina-Conjugadoras , Animales , Humanos , Femenino , Cisplatino/farmacología , Cisplatino/uso terapéutico , MicroARNs/metabolismo , ARN Circular/genética , ARN Circular/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Línea Celular Tumoral , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Helicasas/uso terapéutico , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Ubiquitinación , Proliferación Celular , Resistencia a AntineoplásicosRESUMEN
RNA-binding proteins (RBPs) regulate diverse cellular processes by dynamically interacting with RNA targets. However, effective methods to capture both stable and transient interactions between RBPs and their RNA targets are still lacking, especially when the interaction is dynamic or samples are limited. Here we present an assay of reverse transcription-based RBP binding site sequencing (ARTR-seq), which relies on in situ reverse transcription of RBP-bound RNAs guided by antibodies to identify RBP binding sites. ARTR-seq avoids ultraviolet crosslinking and immunoprecipitation, allowing for efficient and specific identification of RBP binding sites from as few as 20 cells or a tissue section. Taking advantage of rapid formaldehyde fixation, ARTR-seq enables capturing the dynamic RNA binding by RBPs over a short period of time, as demonstrated by the profiling of dynamic RNA binding of G3BP1 during stress granule assembly on a timescale as short as 10 minutes.
Asunto(s)
ARN , Transcripción Reversa , ARN/genética , ARN/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Sitios de Unión/genética , Unión ProteicaRESUMEN
Under various cellular stress conditions, including exposure to toxic chemicals, RNA-binding proteins (RBPs), including Ras GTPase-activating protein-binding protein 1 (G3BP1), aggregate and form stress granule complexes, which serve as hallmarks of cellular stress. The existing methods for analyzing stress granule assembly have limitations in the rapid detection of dynamic cellular stress and ignore the effects of constitutively overexpressed RBP on cellular stress and stress-related processes. Therefore, to overcome these limitations, we established a G3BP1-GFP reporter in a human lung epithelial cell line using CRISPR/Cas9-based knock-in as an alternative system for stress granule analysis. We showed that the G3BP1-GFP reporter system responds to stress conditions and forms a stress granule complex similar to that of native G3BP1. Furthermore, we validated the stress granule response of an established cell line under exposure to various household chemicals. Overall, this novel G3BP1-GFP reporter human lung cell system is capable of monitoring stress granule dynamics in real time and can be used for assessing the lung toxicity of various substances in vitro.
Asunto(s)
ADN Helicasas , Pulmón , ARN Helicasas , Gránulos de Estrés , Humanos , ADN Helicasas/metabolismo , Pulmón/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Gránulos de Estrés/metabolismo , Proteínas Fluorescentes Verdes , Genes ReporterosRESUMEN
Immune checkpoint inhibitors (ICIs) show promise as second-line treatment for advanced bladder cancer (BLCA); however, their responsiveness is limited by the immune evasion mechanisms in tumor cells. This study conduct a Cox regression analysis to screen mRNA-binding proteins and reveals an association between Ras GTPase-activating protein-binding protein 1 (G3BP1) and diminished effectiveness of ICI therapy in patients with advanced BLCA. Subsequent investigation demonstrates that G3BP1 enhances immune evasion in BLCA cells by downregulating major histocompatibility complex class I (MHC-I) through phosphoinositide 3-kinase (PI3K)/Akt signaling activation. Mechanistically, G3BP1 interacts with splicing factor synergistic lethal with U5 snRNA 7 (SLU7) to form a complex with poly(A)-binding protein cytoplasmic 1 and eukaryotic translation initiation factor 4 gamma 1. This complex stabilizes the closed-loop structure of the mRNAs of class IA PI3Ks and consequently facilitates their translation and stabilization, thereby activating PI3K/Akt signaling to downregulate MHC-I. Consistently, targeting G3BP1 with epigallocatechin gallate (EGCG) impedes immune evasion and sensitizes BLCA cells to anti-programmed cell death (PD)-1 antibodies in mice. Thus, G3BP1 and SLU7 collaboratively contribute to immune evasion in BLCA, indicating that EGCG is a precision therapeutic agent to enhance the effectiveness of anti-PD-1 therapy.
Asunto(s)
ADN Helicasas , Neoplasias de la Vejiga Urinaria , Humanos , Animales , Ratones , ADN Helicasas/genética , ADN Helicasas/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Fosfatidilinositol 3-Quinasas , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Evasión Inmune , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas Portadoras/genética , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Factores de Empalme de ARNRESUMEN
The nitrogen-fixing bacteroids inhabit inside legume root nodules must manage finely the utilization of P and Fe, the two most critical elements, due to their antagonistic interactions. While the balance mechanism for them remains unclear. A double SH3 domain-containing protein (dSH3) in the Bradyrhizobium diazoefficiens USDA110 was found to inhibit the alkaline phosphatase activity, thereby reducing P supply from organophosphates. The dSH3 gene is adjacent to the irr gene, which encodes the iron response repressor and regulates Fe homeostasis under Fe-limited conditions. Their transcription directions converge to a common intergenic sequence (IGS) region, forming a convergent transcription. Extending the IGS region through Tn5 transposon or pVO155 plasmid insertion significantly down-regulated expression of this gene pair, leading to a remarkable accumulation of P and an inability to grow under Fe-limited conditions. Inoculation of soybean with either of the insertion mutants resulted in N2-fixing failure. However, the IGS-deleted mutant showed no visible changes in N2-fixing efficiency on soybean compared to that inoculated with wild type. These findings reveal a novel regulative strategy in the IGS region and its flanking convergent gene pair for antagonistic utilization of P and Fe in rhizobia and coordination of N2-fixing efficiency.
Asunto(s)
Proteínas Bacterianas , Bradyrhizobium , Glycine max , Fijación del Nitrógeno , Proteínas de Plantas , Proteínas con Motivos de Reconocimiento de ARN , Bradyrhizobium/genética , Bradyrhizobium/fisiología , Glycine max/microbiología , Homeostasis , Simbiosis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Stress granules (SGs) constitute a signaling hub that plays a critical role in type I interferon responses. Here, we report that growth arrest and DNA damage-inducible beta (Gadd45ß) act as a positive regulator of SG-mediated interferon signaling by targeting G3BP upon RNA virus infection. Gadd45ß deficiency markedly impairs SG formation and SG-mediated activation of interferon signaling in vitro. Gadd45ß knockout mice are highly susceptible to RNA virus infection, and their ability to produce interferon and cytokines is severely impaired. Specifically, Gadd45ß interacts with the RNA-binding domain of G3BP, leading to conformational expansion of G3BP1 via dissolution of its autoinhibitory electrostatic intramolecular interaction. The acidic loop 1- and RNA-binding properties of Gadd45ß markedly increase the conformational expansion and RNA-binding affinity of the G3BP1-Gadd45ß complex, thereby promoting assembly of SGs. These findings suggest a role for Gadd45ß as a component and critical regulator of G3BP1-mediated SG formation, which facilitates RLR-mediated interferon signaling.
Asunto(s)
Interferón Tipo I , Infecciones por Virus ARN , Animales , Ratones , Gránulos Citoplasmáticos/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/genética , Gránulos de EstrésRESUMEN
Background: Alternative splicing (AS) is a biological process that allows genes to be translated into diverse proteins. However, aberrant AS can predispose cells to aberrations in biological mechanisms. RNA binding proteins (RBPs), closely affiliated with AS, have gained increased attention in recent years. Among these RBPs, RBM25 has been reported to participate in the cardiac pathological mechanism through regulating AS; however, the involvement of RBM25 as a splicing factor in heart failure remains unclarified. Methods: RBM25 was overexpressed in H9c2 cells to explore the target genes bound and regulated by RBM25 during heart failure. RNA sequencing (RNA-seq) was used to scrutinize the comprehensive transcriptional level before identifying AS events influenced by RBM25. Further, improved RNA immunoprecipitation sequencing (iRIP-seq) was employed to pinpoint RBM25-binding sites, and RT-qPCR was used to validate specific genes modulated by RBM25. Results: RBM25 was found to upregulate the expression of genes pertinent to the inflammatory response and viral processes, as well as to mediate the AS of genes associated with cellular apoptosis and inflammation. Overlap analysis between RNA-seq and iRIP-seq suggested that RBM25 bound to and manipulated the AS of genes associated with inflammation in H9c2 cells. Moreover, qRT-PCR confirmed Slc38a9, Csf1, and Coro6 as the binding and AS regulatory targets of RBM25. Conclusion: Our research implies that RBM25 plays a contributory role in cardiac inflammatory responses via its ability to bind to and regulate the AS of related genes. This study offers preliminary evidence of the influence of RBM25 on inflammation in H9c2 cells.
Asunto(s)
Empalme Alternativo , Insuficiencia Cardíaca , Proteínas con Motivos de Reconocimiento de ARN , Factores de Empalme de ARN , Empalme Alternativo/genética , Insuficiencia Cardíaca/genética , Inflamación/genética , Proteínas Nucleares/genética , Proteínas de Unión al ARN/genética , Animales , Ratas , Factores de Empalme de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/genéticaRESUMEN
BACKGROUND: Ras-GTPase-activating protein binding protein 1 (G3BP1) is an oncogenic factor, which highly expressed in a variety of cancers. In recent years, G3BP1 has been reported to promote the development of prostate cancer by inhibiting the degradation of AR through inhibiting SPOP. However, whether G3BP1 contributes in a similar manner to the abnormal accumulation of ERα, which is also an important target for hormone therapy, remains unknown. This article addresses this issue and explores potential mechanisms. METHODS: Bioinformatics tools were used for G3BP1 expression analysis, survival analysis, and clinical association analysis. Immunohistochemical staining was used to examine the correlation between G3BP1 and ERα in EC patients. In addition, western blot and co-immunoprecipitation were used to detect the half-life of G3BP1 and mutant, and the effect of G3BP1 and mutant on the ubiquitination and degradation of ERα mediated by SPOP. Then, the oncogenic functions of G3BP1 dependent on the SPOP/ERα axis were determined by CCK8 cell proliferation assay, colony formation assay and cell migration assay. Finally, we established the EC cells treated or untreated with fulvestrant, exploring the possibility of fulvestrant combined with the reduction of G3BP1 to improve the efficacy of fulvestrant. RESULTS: G3BP1 is abnormally high expressed and characterized by high-frequency mutation in EC. In addition, there is a positive correlation between G3BP1 protein and ERα protein. Mechanistically, both G3BP1 and mutant, the latter is displaying the longer half-life, competitively bind SPOP with ERα, thereby inhibiting SPOP-mediated ubiquitination and degradation of ERα. Functionally, G3BP1 and mutant promote the proliferation and migration of EC cells by regulating the G3BP1/SPOP/ERα axis. However, fulvestrant can reverse the cancer-promoting effects of G3BP1 and mutant. CONCLUSIONS: G3BP1 and its mutant positively regulate ERα signaling pathway by inhibiting SPOP-mediated ubiquitination and degradation of ERα, indicating the promising effect of fulvestrant on the suppression the occurrence and development of EC with high expressed G3BP1 and G3BP1 mutants. Video Abstract.
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Neoplasias Endometriales , Receptor alfa de Estrógeno , Femenino , Humanos , Masculino , Transformación Celular Neoplásica , ADN Helicasas/genética , ADN Helicasas/metabolismo , Neoplasias Endometriales/metabolismo , Receptor alfa de Estrógeno/metabolismo , Fulvestrant , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , UbiquitinaciónRESUMEN
RNA-binding proteins (RBPs) are the proteins that bind RNAs and regulate their functioning. RBPs in mosquitoes are gaining attention due to their ability to bind flaviviruses and regulate their replication and transmission. Despite their relevance, RBPs in mosquitoes are not explored much. In this study, we screened the whole genome of Aedes aegypti, the primary vector of several pathogenic viruses, and identified the proteins containing RNA recognition motif (RRM), the most abundant protein domain in eukaryotes. Using several in silico strategies, a total of 135 RRM-containing RBPs were identified in Ae. aegypti. The proteins were characterized based on their available annotations and the sequence similarity with Drosophila melanogaster. Ae. aegypti RRM-containing RBPs included serine/arginine-rich (SR) proteins, polyadenylate-binding proteins (PABP), heteronuclear ribonucleoproteins (hnRNP), small nuclear ribonucleoproteins (snRNP), splicing factors, eukaryotic initiation factors, transformers, and nucleolysins. Phylogenetic analysis revealed that the proteins and the domain organization are conserved among Ae. aegypti, Bombyx mori, and Drosophila melanogaster. However, the gene length and the intron-exon organization varied across the insect species. Expression analysis of the genes encoding RBPs using publicly available RNA sequencing data for different developmental time points of the mosquito life cycle starting from the ovary and eggs up to the adults revealed stage-specific expression with several genes preferentially expressed in early embryonic stages and blood-fed female ovaries. This is the first database for the Ae. aegypti RBPs that can serve as the reference base for future investigations. Stage-specific genes can be further explored to determine their role in mosquito growth and development with a focus on developing novel mosquito control strategies.
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Aedes , Animales , Femenino , Aedes/fisiología , Proteínas con Motivos de Reconocimiento de ARN/genética , Drosophila melanogaster/genética , Filogenia , Motivo de Reconocimiento de ARN , Mosquitos Vectores , Proteínas de Unión al ARN/genética , ARNRESUMEN
The nucleocapsid protein N of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enwraps and condenses the viral genome for packaging but is also an antagonist of the innate antiviral defense. It suppresses the integrated stress response (ISR), purportedly by interacting with stress granule (SG) assembly factors G3BP1 and 2, and inhibits type I interferon responses. To elucidate its mode of action, we systematically deleted and over-expressed distinct regions and domains. We show that N via domain N2b blocks PKR-mediated ISR activation, as measured by suppression of ISR-induced translational arrest and SG formation. N2b mutations that prevent dsRNA binding abrogate these activities also when introduced in the intact N protein. Substitutions reported to block post-translation modifications of N or its interaction with G3BP1/2 did not have a detectable additive effect. In an encephalomyocarditis virus-based infection model, N2b - but not a derivative defective in RNA binding-prevented PKR activation, inhibited ß-interferon expression and promoted virus replication. Apparently, SARS-CoV-2 N inhibits innate immunity by sequestering dsRNA to prevent activation of PKR and RIG-I-like receptors. Similar observations were made for the N protein of human coronavirus 229E, suggesting that this may be a general trait conserved among members of other orthocoronavirus (sub)genera.