RESUMEN

Heat stress (HS) poses a significant challenge to plant survival, necessitating sophisticated molecular mechanisms to maintain cellular homeostasis. Here, we identify SICKLE (SIC) as a key modulator of HS responses in Arabidopsis (Arabidopsis thaliana). SIC is required for the sequestration of RNA DEBRANCHING ENZYME 1 (DBR1), a rate-limiting enzyme of lariat intronic RNA (lariRNA) decay, into stress granules (SGs). The sequestration of DBR1 by SIC enhances the accumulation of lariRNAs, branched circular RNAs derived from excised introns during pre-mRNA splicing, which in turn promote the transcription of their parental genes. Our findings further demonstrate that SIC-mediated DBR1 sequestration in SGs is crucial for plant HS tolerance, as deletion of the N-terminus of SIC (SIC1-244) impairs DBR1 sequestration and compromises plant response to HS. Overall, our study unveils a mechanism of transcriptional regulation in the HS response, where lariRNAs are enriched through DBR1 sequestration, ultimately promoting the transcription of heat stress tolerance genes.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis , Regulación de la Expresión Génica de las Plantas , Respuesta al Choque Térmico , Intrones , Empalme del ARN , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Respuesta al Choque Térmico/genética , Intrones/genética , Gránulos de Estrés/metabolismo , Gránulos de Estrés/genética , ARN de Planta/metabolismo , ARN de Planta/genética , Termotolerancia/genética , ARN Circular/metabolismo , ARN Circular/genética , Plantas Modificadas Genéticamente

RESUMEN

Stress granules (SGs) are conserved reversible cytoplasmic condensates enriched with aggregation-prone proteins assembled in response to various stresses. How plants regulate SG dynamics is unclear. Here, we show that 26S proteasome is a stable component of SGs, promoting the overall clearance of SGs without affecting the molecular mobility of SG components. Increase in either temperature or duration of heat stress reduces the molecular mobility of SG marker proteins and suppresses SG clearance. Heat stress induces dramatic ubiquitylation of SG components and enhances the activities of SG-resident proteasomes, allowing the degradation of SG components even during the assembly phase. Their proteolytic activities enable the timely disassembly of SGs and secure the survival of plant cells during the recovery from heat stress. Therefore, our findings identify the cellular process that de-couples macroscopic dynamics of SGs from the molecular dynamics of its constituents and highlights the significance of the proteasomes in SG disassembly.

Asunto(s)

Arabidopsis , Respuesta al Choque Térmico , Complejo de la Endopetidasa Proteasomal , Ubiquitinación , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/enzimología , Proteolisis , Gránulos de Estrés/metabolismo , Gránulos de Estrés/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Gránulos Citoplasmáticos/metabolismo

RESUMEN

Stress granules (SGs) are cytoplasmic messenger ribonucleoprotein granules transiently formed in stressed mammalian cells. Although SG components have been well characterized, detailed insights into the molecular behavior inside SGs remain unresolved. We investigated nanoscale dynamics and localization of endogenous mRNAs in SGs combining single mRNA tracking and super-resolution localization microscopy. First, we developed a methodology for tracking single mRNAs within SGs, revealing that although mRNAs in SGs are mainly stationary (∼40%), they also move in a confined (∼25%) or freely diffusing (∼35%) manner. Second, the super-resolution localization microscopy showed that the mRNAs in SGs are heterogeneously distributed and partially form high-density clusters. Third, we simultaneously performed single mRNA tracking and super-resolution microscopy in SGs, demonstrating that single mRNA trajectories are mainly found around high-density clusters. Finally, a quantitative analysis of mRNA localization and dynamics during stress removal was conducted using live super-resolution imaging and single-molecule tracking. These results suggest that SGs have a highly organized structure that enables dynamic regulation of the mRNAs at the nanoscale, which is responsible for the ordered formation and the wide variety of functions of SGs.

Asunto(s)

ARN Mensajero , Imagen Individual de Molécula , Gránulos de Estrés , ARN Mensajero/metabolismo , ARN Mensajero/genética , Humanos , Imagen Individual de Molécula/métodos , Gránulos de Estrés/metabolismo , Gránulos de Estrés/genética , Células HeLa , Transporte de ARN , Gránulos Citoplasmáticos/metabolismo , Microscopía Fluorescente/métodos

RESUMEN

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.

Asunto(s)

Citoplasma , Polirribosomas , Ribonucleoproteínas , Polirribosomas/metabolismo , Citoplasma/metabolismo , Humanos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Simulación de Dinámica Molecular , ARN Mensajero/metabolismo , ARN Mensajero/genética , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Condensados Biomoleculares/metabolismo , Gránulos de Estrés/metabolismo , Gránulos de Estrés/genética

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-Oncogénicas

RESUMEN

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.

Asunto(s)

Proteína 1 de la Respuesta de Crecimiento Precoz , Lomustina , ARN Mensajero , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Lomustina/farmacología , Gránulos de Estrés/metabolismo , Gránulos de Estrés/genética , Apoptosis/efectos de los fármacos , Antineoplásicos Alquilantes/farmacología

RESUMEN

Stress induces global stabilization of the mRNA poly(A) tail (PAT) and the assembly of untranslated poly(A)-tailed mRNA into mRNPs that accumulate in stress granules (SGs). While the mechanism behind stress-induced global PAT stabilization has recently emerged, the biological significance of PAT stabilization under stress remains elusive. Here, we demonstrate that stress-induced PAT stabilization is a prerequisite for SG formation. Perturbations in PAT length impact SG formation; PAT shortening, achieved by overexpressing mRNA deadenylases, inhibits SG formation, whereas PAT lengthening, achieved by overexpressing their dominant negative mutants or downregulating deadenylases, promotes it. PABPC1, which specifically binds to the PAT, is crucial for SG formation. Complementation analyses reveal that the PABC/MLLE domain of PABPC1, responsible for binding PAM2 motif-containing proteins, plays a key role. Among them, ataxin-2 is a known SG component. A dominant-negative approach reveals that the PAM2 motif of ataxin-2 is essential for SG formation. Notably, ataxin-2 increases stress sensitivity, lowering the threshold for SG formation, probably by promoting the aggregation of PABPC1-bound mRNA. The C-terminal region is responsible for the self-aggregation of ataxin-2. These findings underscore the critical roles of mRNA PAT, PABPC1 and ataxin-2 in SG formation and provide mechanistic insights into this process.

Asunto(s)

Ataxina-2 , Poli A , Proteína I de Unión a Poli(A) , ARN Mensajero , Gránulos de Estrés , Proteína I de Unión a Poli(A)/metabolismo , Proteína I de Unión a Poli(A)/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Ataxina-2/metabolismo , Ataxina-2/genética , Humanos , Gránulos de Estrés/metabolismo , Gránulos de Estrés/genética , Poli A/metabolismo , Unión Proteica , Estabilidad del ARN , Células HeLa , Estrés Fisiológico/genética

RESUMEN

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.

Asunto(s)

Senescencia Celular , Cromatina , Metiltransferasas , Gránulos de Estrés , Metiltransferasas/metabolismo , Metiltransferasas/genética , Cromatina/metabolismo , Humanos , Gránulos de Estrés/metabolismo , Gránulos de Estrés/genética , Hexoquinasa/metabolismo , Hexoquinasa/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Adenosina/metabolismo , Adenosina/análogos & derivados , Células HEK293 , Reprogramación Metabólica , Separación de Fases

RESUMEN

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.

Asunto(s)

Estrés del Retículo Endoplásmico , Endorribonucleasas , Proteínas Serina-Treonina Quinasas , Proteína 1 de Unión a la X-Box , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Endorribonucleasas/metabolismo , Endorribonucleasas/genética , Humanos , Proteína 1 de Unión a la X-Box/metabolismo , Proteína 1 de Unión a la X-Box/genética , Animales , Empalme del ARN , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/genética , Gránulos de Estrés/metabolismo , Gránulos de Estrés/genética , Factores de Transcripción del Factor Regulador X/metabolismo , Factores de Transcripción del Factor Regulador X/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Respuesta de Proteína Desplegada , Ratones , Células HeLa , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/genética , Transducción de Señal

RESUMEN

Porcine epidemic diarrhea virus (PEDV) envelope protein (E) has been characterized as an important structural protein that plays critical roles in the interplay with its host to affect the virus life cycle. Stress granules (SGs) are host translationally silent ribonucleoproteins, which are mainly induced by the phosphorylation of eIF2α in the PERK/eIF2α signaling pathway. Our previous study found that PEDV E protein caused endoplasmic reticulum stress response (ERS)-mediated suppression of antiviral proteins' translation. However, the link and the underlying mechanism by which PEDV induces SGs formation and suppresses host translation remain elusive. In this study, our results showed that PEDV E protein significantly elevated the expression of GRP78, CANX, and phosphorylation of PERK and eIF2α, indicating that the PERK/eIF2α branch of ERS was activated. PEDV E protein localized to the ER and aggregated into puncta to reconstruct ER structure, and further induced SGs formation, which has been caused through upregulating the G3BP1 expression level. In addition, a significant global translational stall and endogenous protein translation attenuation were detected in the presence of E protein overexpression, but the global mRNA transcriptional level remained unchanged, suggesting that the shutoff of protein translation was associated with the translation, not with the transcription process. Collectively, this study demonstrates that PERK/eIF2α activation is required for SGs formation and protein translation stall. This study is beneficial for us to better understand the mechanism by which PEDV E suppresses host protein synthesis, and provides us a new insight into the host translation regulation during virus infection.

Asunto(s)

Factor 2 Eucariótico de Iniciación , Virus de la Diarrea Epidémica Porcina , Biosíntesis de Proteínas , Transducción de Señal , Gránulos de Estrés , Proteínas del Envoltorio Viral , eIF-2 Quinasa , Animales , Chlorocebus aethiops , eIF-2 Quinasa/metabolismo , eIF-2 Quinasa/genética , Chaperón BiP del Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Fosforilación , Virus de la Diarrea Epidémica Porcina/fisiología , Gránulos de Estrés/metabolismo , Gránulos de Estrés/genética , Porcinos , Células Vero , Proteínas del Envoltorio Viral/metabolismo

RESUMEN

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/genética

RESUMEN

T-cell intracellular antigen-1 (TIA-1) is a key RNA-binding protein that participates in translation regulation and RNA splicing. TIA-1 undergoes liquid-liquid phase separation as a fundamental mechanism that enables the condensation of RNA and proteins into membraneless organelles called stress granules (SGs). However, this dynamic behavior can lead to aberrant fibril formation, implicated in neurodegenerative disorders, and must be tightly regulated. In this study, we investigated the role in the cell of histidine residues His94 and His96, responsible for Zn2+ binding. Using fluorescence microscopy, we found that the specific binding site formed by these residues is critical for SG assembly. Furthermore, it also plays a role maintaining the dynamic behavior of SG-assembled TIA-1. Collectively, our findings confirm the physiological relevance of TIA-1 His94 and His96 in the Zn2+-mediated regulatory mechanism for protection against fibril formation in SGs.

Asunto(s)

Histidina , Gránulos de Estrés , Antígeno Intracelular 1 de las Células T , Zinc , Antígeno Intracelular 1 de las Células T/metabolismo , Antígeno Intracelular 1 de las Células T/genética , Zinc/metabolismo , Histidina/metabolismo , Histidina/genética , Histidina/química , Gránulos de Estrés/metabolismo , Gránulos de Estrés/genética , Humanos , Unión Proteica , Sitios de Unión , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/genética

RESUMEN

The assembly of membrane-less organelles such as stress granules (SGs) is emerging as central in helping cells rapidly respond and adapt to stress. Following stress sensing, the resulting global translational shutoff leads to the condensation of stalled mRNAs and proteins into SGs. By reorganizing cytoplasmic contents, SGs can modulate RNA translation, biochemical reactions, and signaling cascades to promote survival until the stress is resolved. While mechanisms for SG disassembly are not widely understood, the resolution of SGs is important for maintaining cell viability and protein homeostasis. Mutations that lead to persistent or aberrant SGs are increasingly associated with neuropathology and a hallmark of several neurodegenerative diseases. Mutations in CLN3 are causative of juvenile neuronal ceroid lipofuscinosis, a rare neurodegenerative disease affecting children also known as Batten disease. CLN3 encodes a transmembrane lysosomal protein implicated in autophagy, endosomal trafficking, metabolism, and response to oxidative stress. Using a HeLa cell model lacking CLN3, we now show that CLN3KO is associated with an altered metabolic profile, reduced global translation, and altered stress signaling. Furthermore, loss of CLN3 function results in perturbations in SG dynamics, resulting in assembly and disassembly defects, and altered expression of the key SG nucleating factor G3BP1. With a growing interest in SG-modulating drugs for the treatment of neurodegenerative diseases, novel insights into the molecular basis of CLN3 Batten disease may reveal avenues for disease-modifying treatments for this debilitating childhood disease.

Asunto(s)

Expresión Génica , Chaperonas Moleculares , Lipofuscinosis Ceroideas Neuronales , Gránulos de Estrés , Humanos , Células HeLa , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Lipofuscinosis Ceroideas Neuronales/genética , Lipofuscinosis Ceroideas Neuronales/fisiopatología , Gránulos de Estrés/genética , Gránulos de Estrés/patología , Estrés Fisiológico/genética , Transducción de Señal/genética , Expresión Génica/genética , Línea Celular

RESUMEN

Rationale: A C9orf72 hexanucleotide repeat expansion (GGGGCC) is the most common genetic origin of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Haploinsufficiency of C9orf72 has been proposed as a possible disease mechanism (loss-of-function mechanism). Additionally, the aberrantly activated unfolded protein response (UPR) and stress granule (SG) formation are associated with the etiopathology of both ALS and FTD. However, the molecular determinants in this pathogenesis are not well characterized. Methods: We performed an immunoprecipitation-mass spectrometry (IP-MS) assay to identify potential proteins interacting with the human C9orf72 protein. We used C9orf72 knockout cell and rat models to determine the roles of C9orf72 in translation initiation and the stress response. Results: Here, we show that C9orf72, which is genetically and pathologically related to ALS and FTD, interacts with eukaryotic initiation factor 2 subunit alpha (eIF2α) and regulates its function in translation initiation. C9orf72 knockout weakens the interaction between eIF2α and eIF2B5, leading to global translation inhibition. Moreover, the loss of C9orf72 results in primary ER stress with activated UPR in rat spleens, which is one of the causes of splenomegaly with inflammation in C9orf72 -/- rats. Finally, C9orf72 delays SG formation by interacting with eIF2α in stressed cells. Conclusions: In summary, these data reveal that C9orf72 modulates translation initiation, the UPR and SG formation, which have implications for understanding ALS/FTD pathogenesis.

Asunto(s)

Esclerosis Amiotrófica Lateral , Proteína C9orf72 , Demencia Frontotemporal , Animales , Humanos , Ratas , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Expansión de las Repeticiones de ADN , Factor 2 Eucariótico de Iniciación/genética , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Gránulos de Estrés/genética , Gránulos de Estrés/metabolismo , Respuesta de Proteína Desplegada/genética , Respuesta de Proteína Desplegada/fisiología

RESUMEN

Stress granules (SGs) are dynamic structures that store cytosolic messenger ribonucleoproteins. SGs have recently been shown to serve as a platform for activating antiviral innate immunity; however, several pathogenic viruses suppress SG formation to evade innate immunity. In this study, we investigated the relationship between rabies virus (RABV) virulence and SG formation, using viral strains with different levels of virulence. We found that the virulent Nishigahara strain did not induce SG formation, but its avirulent offshoot, the Ni-CE strain, strongly induced SG formation. Furthermore, we demonstrated that the amino acid at position 95 in the RABV matrix protein (M95), a pathogenic determinant for the Nishigahara strain, plays a key role in inhibiting SG formation, followed by protein kinase R (PKR)-dependent phosphorylation of the α subunit of eukaryotic initiation factor 2α (eIF2α). M95 was also implicated in the accumulation of RIG-I, a viral RNA sensor protein, in SGs and in the subsequent acceleration of interferon induction. Taken together, our findings strongly suggest that M95-related inhibition of SG formation contributes to the pathogenesis of RABV by allowing the virus to evade the innate immune responses of the host. IMPORTANCE Rabies virus (RABV) is a neglected zoonotic pathogen that causes lethal infections in almost all mammalian hosts, including humans. Recently, RABV has been reported to induce intracellular formation of stress granules (SGs), also known as platforms that activate innate immune responses. However, the relationship between SG formation capacity and pathogenicity of RABV has remained unclear. In this study, by comparing two RABV strains with completely different levels of virulence, we found that the amino acid mutation from valine to alanine at position 95 of matrix protein (M95), which is known to be one of the amino acid mutations that determine the difference in virulence between the strains, plays a major role in SG formation. Importantly, M95 was involved in the accumulation of RIG-I in SGs and in promoting interferon induction. These findings are the first report of the effect of a single amino acid substitution associated with SGs on viral virulence.

Asunto(s)

Virus de la Rabia , Gránulos de Estrés , Proteínas de la Matriz Viral , Aminoácidos/metabolismo , Animales , Factor 2 Eucariótico de Iniciación/metabolismo , Humanos , Interferones/inmunología , Proteínas Quinasas/inmunología , ARN Viral/metabolismo , Virus de la Rabia/genética , Virus de la Rabia/patogenicidad , Ribonucleoproteínas/metabolismo , Gránulos de Estrés/genética , Gránulos de Estrés/inmunología , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/inmunología , Proteínas Virales/genética , Proteínas Virales/metabolismo

RESUMEN

Obesity is a global epidemic and a major predisposing factor for cancer. Increasing evidence shows that obesity-associated stress is a key driver of cancer risk and progression. Previous work has identified the phase-separation organelles, stress granules (SG), as mutant KRAS-dependent mediators of stress adaptation. However, the dependence of tumorigenesis on these organelles is unknown. Here, we establish a causal link between SGs and pancreatic ductal adenocarcinoma (PDAC). Importantly, we uncover that dependence on SGs is drastically heightened in obesity-associated PDAC. Furthermore, we identify a previously unknown regulator and component of SGs, namely, the serine/arginine protein kinase 2 (SRPK2), as a specific determinant of SG formation in obesity-associated PDAC. We show that SRPK2-mediated SG formation in obesity-associated PDAC is driven by hyperactivation of the IGF1/PI3K/mTOR/S6K1 pathway and that S6K1 inhibition selectively attenuates SGs and impairs obesity-associated PDAC development. SIGNIFICANCE: : We show that stress adaptation via the phase-separation organelles SGs mediates PDAC development. Moreover, preexisting stress conditions such as obesity are a driving force behind tumor SG dependence, and enhanced SG levels are key determinants and a chemopreventive target for obesity-associated PDAC. This article is highlighted in the In This Issue feature, p. 1825.

Asunto(s)

Carcinoma Ductal Pancreático , Obesidad , Neoplasias Pancreáticas , Proteínas Serina-Treonina Quinasas , Gránulos de Estrés , Carcinoma Ductal Pancreático/etiología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Humanos , Obesidad/complicaciones , Obesidad/genética , Obesidad/metabolismo , Neoplasias Pancreáticas/etiología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Gránulos de Estrés/genética , Gránulos de Estrés/metabolismo , Neoplasias Pancreáticas

RESUMEN

As a highly pathogenic human coronavirus, SARS-CoV-2 has to counteract an intricate network of antiviral host responses to establish infection and spread. The nucleic acid-induced stress response is an essential component of antiviral defense and is closely related to antiviral innate immunity. However, whether SARS-CoV-2 regulates the stress response pathway to achieve immune evasion remains elusive. In this study, SARS-CoV-2 NSP5 and N protein were found to attenuate antiviral stress granule (avSG) formation. Moreover, NSP5 and N suppressed IFN expression induced by infection of Sendai virus or transfection of a synthetic mimic of dsRNA, poly (I:C), inhibiting TBK1 and IRF3 phosphorylation, and restraining the nuclear translocalization of IRF3. Furthermore, HEK293T cells with ectopic expression of NSP5 or N protein were less resistant to vesicular stomatitis virus infection. Mechanistically, NSP5 suppressed avSG formation and disrupted RIG-I-MAVS complex to attenuate the RIG-I-mediated antiviral immunity. In contrast to the multiple targets of NSP5, the N protein specifically targeted cofactors upstream of RIG-I. The N protein interacted with G3BP1 to prevent avSG formation and to keep the cofactors G3BP1 and PACT from activating RIG-I. Additionally, the N protein also affected the recognition of dsRNA by RIG-I. This study revealed the intimate correlation between SARS-CoV-2, the stress response, and innate antiviral immunity, shedding light on the pathogenic mechanism of COVID-19.

Asunto(s)

Proteasas 3C de Coronavirus/genética , Proteínas de la Nucleocápside de Coronavirus/genética , Proteína 58 DEAD Box/genética , ADN Helicasas/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Helicasas/genética , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas de Unión al ARN/genética , Receptores Inmunológicos/genética , SARS-CoV-2/genética , Gránulos de Estrés/genética , Animales , Chlorocebus aethiops , Proteasas 3C de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteína 58 DEAD Box/inmunología , ADN Helicasas/inmunología , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Evasión Inmune , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Poli I-C/farmacología , Proteínas de Unión a Poli-ADP-Ribosa/inmunología , Unión Proteica , ARN Helicasas/inmunología , Proteínas con Motivos de Reconocimiento de ARN/inmunología , ARN Bicatenario/genética , ARN Bicatenario/inmunología , Proteínas de Unión al ARN/inmunología , Receptores Inmunológicos/inmunología , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Virus Sendai/genética , Virus Sendai/inmunología , Transducción de Señal , Gránulos de Estrés/efectos de los fármacos , Gránulos de Estrés/inmunología , Gránulos de Estrés/virología , Células Vero , Vesiculovirus/genética , Vesiculovirus/inmunología

RESUMEN

Flaviviruses are human pathogens that can cause severe diseases, such as dengue fever and Japanese encephalitis, which can lead to death. Valosin-containing protein (VCP)/p97, a cellular ATPase associated with diverse cellular activities (AAA-ATPase), is reported to have multiple roles in flavivirus replication. Nevertheless, the importance of each role still has not been addressed. In this study, the functions of 17 VCP mutants that are reportedly unable to interact with the VCP cofactors were validated using the short-interfering RNA rescue experiments. Our findings of this study suggested that VCP exerts its functions in replication of the Japanese encephalitis virus by interacting with the VCP cofactor nuclear protein localization 4 (NPL4). We show that the depletion of NPL4 impaired the early stage of viral genome replication. In addition, we demonstrate that the direct interaction between NPL4 and viral nonstructural protein (NS4B) is critical for the translocation of NS4B to the sites of viral replication. Finally, we found that Japanese encephalitis virus and dengue virus promoted stress granule formation only in VCP inhibitor-treated cells and the expression of NS4B or VCP attenuated stress granule formation mediated by protein kinase R, which is generally known to be activated by type I interferon and viral genome RNA. These results suggest that the NS4B-mediated recruitment of VCP to the virus replication site inhibits cellular stress responses and consequently facilitates viral protein synthesis in the flavivirus-infected cells.

Asunto(s)

Virus de la Encefalitis Japonesa (Especie) , Flavivirus , Proteínas Nucleares , Gránulos de Estrés , Proteína que Contiene Valosina , Proteínas no Estructurales Virales , Replicación Viral , Virus de la Encefalitis Japonesa (Especie)/genética , Virus de la Encefalitis Japonesa (Especie)/metabolismo , Virus de la Encefalitis Japonesa (Especie)/fisiología , Flavivirus/genética , Flavivirus/metabolismo , Flavivirus/fisiología , Genoma Viral , Humanos , Proteínas Nucleares/metabolismo , ARN Viral/genética , Gránulos de Estrés/genética , Gránulos de Estrés/metabolismo , Proteína que Contiene Valosina/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/fisiología

RESUMEN

Ribonucleoprotein granules are ubiquitous features of eukaryotic cells. Several observations argue that the formation of at least some RNP granules can be considered analogous to the formation of unfolded protein aggregates. First, unfolded protein aggregates form from the exposure of promiscuous protein interaction surfaces, while some mRNP granules form, at least in part, by promiscuous intermolecular RNA-RNA interactions due to exposed RNA surfaces when mRNAs are not engaged with ribosomes. Second, analogous to the role of protein chaperones in preventing misfolded protein aggregation, cells contain abundant "RNA chaperones" to limit inappropriate RNA-RNA interactions and prevent mRNP granule formation. Third, analogous to the role of protein aggregates in diseases, situations where RNA aggregation exceeds the capacity of RNA chaperones to disaggregate RNAs may contribute to human disease. Understanding that RNP granules can be considered as promiscuous, reversible RNA aggregation events allow insight into their composition and how cells have evolved functions for RNP granules.

Asunto(s)

Condensados Biomoleculares/química , Proteínas de Choque Térmico/química , Chaperonas Moleculares/química , ARN Mensajero/química , Ribonucleoproteínas/química , Gránulos de Estrés/química , Condensados Biomoleculares/metabolismo , Eucariontes , Células Eucariotas/metabolismo , Factor 4A Eucariótico de Iniciación/química , Factor 4A Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Floculación , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Agregado de Proteínas , Pliegue de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Gránulos de Estrés/genética , Gránulos de Estrés/metabolismo

RESUMEN

Cells have to deal with conditions that can cause damage to biomolecules and eventually cell death. To protect against these adverse conditions and promote recovery, cells undergo dramatic changes upon exposure to stress. This involves activation of signaling pathways, cell cycle arrest, translational reprogramming, and reorganization of the cytoplasm. Notably, many stress conditions cause a global inhibition of mRNA translation accompanied by the formation of cytoplasmic condensates called stress granules (SGs), which sequester mRNA together with RNA-binding proteins, translation initiation factors, and other components. SGs are highly conserved in eukaryotes, suggesting that they perform an important function during the stress response. Over the years, many different roles have been assigned to SGs, including translational control, mRNA storage, regulation of mRNA decay, antiviral innate immune response, and modulation of signaling pathways. Most of our understanding, however, has been deduced from correlative data based upon the composition of SGs and only recently have technological innovations allowed hypotheses for SG function to be directly tested. Here, we discuss these challenges and explore the evidence related to the function of SGs.

Asunto(s)

Gránulos Citoplasmáticos/genética , Inmunidad Innata/genética , ARN Mensajero/genética , Gránulos de Estrés/genética , Gránulos Citoplasmáticos/inmunología , Respuesta al Choque Térmico/genética , Humanos , Estrés Oxidativo/genética , Estabilidad del ARN/genética , Estabilidad del ARN/inmunología , Gránulos de Estrés/inmunología