RESUMEN
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.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Proteínas con Motivos de Reconocimiento de ARN , Gránulos de Estrés , Animales , Ratones , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/metabolismo , Enfermedad de Charcot-Marie-Tooth/patología , Citoplasma , Neuronas Motoras , Proteínas con Motivos de Reconocimiento de ARN/metabolismoRESUMEN
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.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Ribonucleoproteínas/metabolismo , Línea Celular Tumoral , Citoplasma/metabolismo , Estructuras Citoplasmáticas/metabolismo , Células HEK293 , Humanos , Fosforilación , ARN/metabolismoRESUMEN
Defects in nucleocytoplasmic transport have been identified as a key pathogenic event in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) mediated by a GGGGCC hexanucleotide repeat expansion in C9ORF72, the most common genetic cause of ALS/FTD. Furthermore, nucleocytoplasmic transport disruption has also been implicated in other neurodegenerative diseases with protein aggregation, suggesting a shared mechanism by which protein stress disrupts nucleocytoplasmic transport. Here, we show that cellular stress disrupts nucleocytoplasmic transport by localizing critical nucleocytoplasmic transport factors into stress granules, RNA/protein complexes that play a crucial role in ALS pathogenesis. Importantly, inhibiting stress granule assembly, such as by knocking down Ataxin-2, suppresses nucleocytoplasmic transport defects as well as neurodegeneration in C9ORF72-mediated ALS/FTD. Our findings identify a link between stress granule assembly and nucleocytoplasmic transport, two fundamental cellular processes implicated in the pathogenesis of C9ORF72-mediated ALS/FTD and other neurodegenerative diseases.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Esclerosis Amiotrófica Lateral/patología , Ataxina-2/metabolismo , Proteína C9orf72/genética , Demencia Frontotemporal/patología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Anciano , Esclerosis Amiotrófica Lateral/metabolismo , Arsenitos/toxicidad , Ataxina-2/antagonistas & inhibidores , Ataxina-2/genética , Proteína C9orf72/metabolismo , Expansión de las Repeticiones de ADN/genética , Femenino , Demencia Frontotemporal/metabolismo , Células HEK293 , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Persona de Mediana Edad , Proteínas de Complejo Poro Nuclear/metabolismo , Estrés Oxidativo/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Compuestos de Sodio/toxicidad , alfa Carioferinas/antagonistas & inhibidores , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , beta Carioferinas/antagonistas & inhibidores , beta Carioferinas/genética , beta Carioferinas/metabolismo , Proteína de Unión al GTP ran/antagonistas & inhibidores , Proteína de Unión al GTP ran/genética , Proteína de Unión al GTP ran/metabolismoRESUMEN
The heat shock response is crucial for cell survival. In this issue of Molecular Cell, Desroches Altamirano et al.1 demonstrate that a temperature-induced conformational change in the translation initiation factor eIF4G is a key mechanism regulating translation during the heat shock response.
Asunto(s)
Factor 4G Eucariótico de Iniciación , Respuesta al Choque Térmico , Biosíntesis de Proteínas , ARN Mensajero , Factor 4G Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Humanos , Animales , Conformación Proteica , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genéticaRESUMEN
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.
Asunto(s)
Gránulos Citoplasmáticos/genética , ADN Helicasas/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , Biosíntesis de Proteínas , ARN Helicasas/genética , Proteínas con Motivos de Reconocimiento de ARN/genética , Estrés Fisiológico/genética , Regiones no Traducidas 5' , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/patología , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ADN Helicasas/metabolismo , Femenino , Células HCT116 , Células HeLa , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Espermatogonias/citología , Espermatogonias/patología , Testículo/citología , Testículo/metabolismoRESUMEN
Rearranged during transfection (RET) rearrangement oncoprotein-mediated Ras/MAPK signaling cascade is constitutively activated in cancers. Here, we demonstrate a unique signal niche. The niche is a ternary complex based on the chimeric RET liquid-liquid phase separation. The complex comprises the rearranged kinase (RET fusion); the adaptor (GRB2), and the effector (SHC1). Together, they orchestrate the Ras/MAPK signal cascade, which is dependent on tyrosine kinase. CCDC6-RET fusion undergoes LLPS requiring its kinase domain and its fusion partner. The CCDC6-RET fusion LLPS promotes the autophosphorylation of RET fusion, with enhanced kinase activity, which is necessary for the formation of the signaling niche. Within the signal niche, the interactions among the constituent components are reinforced, and the signal transduction efficiency is amplified. The specific RET fusion-related signal niche elucidates the mechanism of the constitutive activation of the Ras/MAPK signaling pathway. Beyond just focusing on RET fusion itself, exploration of the ternary complex potentially unveils a promising avenue for devising therapeutic strategies aimed at treating RET fusion-driven diseases.
Asunto(s)
Proteína Adaptadora GRB2 , Sistema de Señalización de MAP Quinasas , Proteínas de Fusión Oncogénica , Proteínas Proto-Oncogénicas c-ret , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Proteínas ras , Humanos , Proteína Adaptadora GRB2/metabolismo , Proteína Adaptadora GRB2/genética , Células HEK293 , Proteínas de Fusión Oncogénica/metabolismo , Proteínas de Fusión Oncogénica/genética , Fosforilación , Proteínas Proto-Oncogénicas c-ret/metabolismo , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas ras/metabolismo , Proteínas ras/genética , Transducción de Señal , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/genéticaRESUMEN
The molecular understanding of autophagy has originated almost exclusively from yeast genetic studies. Little is known about essential autophagy components specific to higher eukaryotes. Here we perform genetic screens in C. elegans and identify four metazoan-specific autophagy genes, named epg-2, -3, -4, and -5. Genetic analysis reveals that epg-2, -3, -4, and -5 define discrete genetic steps of the autophagy pathway. epg-2 encodes a coiled-coil protein that functions in specific autophagic cargo recognition. Mammalian homologs of EPG-3/VMP1, EPG-4/EI24, and EPG-5/mEPG5 are essential for starvation-induced autophagy. VMP1 regulates autophagosome formation by controlling the duration of omegasomes. EI24 and mEPG5 are required for formation of degradative autolysosomes. This study establishes C. elegans as a multicellular genetic model to delineate the autophagy pathway and provides mechanistic insights into the metazoan-specific autophagic process.
Asunto(s)
Autofagia , Caenorhabditis elegans/genética , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/embriología , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/genética , Gránulos Citoplasmáticos/metabolismo , Lisosomas/metabolismo , Mutación , Fagosomas/metabolismoRESUMEN
How autophagy, an evolutionarily conserved intracellular catabolic system for bulk degradation, selectively degrades protein aggregates is poorly understood. Here, we show that several maternally derived germ P granule components are selectively eliminated by autophagy in somatic cells during C. elegans embryogenesis. The activity of sepa-1 is required for the degradation of these P granule components and for their accumulation into aggregates, termed PGL granules, in autophagy mutants. SEPA-1 forms protein aggregates and is also a preferential target of autophagy. SEPA-1 directly binds to the P granule component PGL-3 and also to the autophagy protein LGG-1/Atg8. SEPA-1 aggregates consistently colocalize with PGL granules and with LGG-1 puncta. Thus, SEPA-1 functions as a bridging molecule in mediating the specific recognition and degradation of P granule components by autophagy. Our study reveals a mechanism for preferential degradation of protein aggregates by autophagy and emphasizes the physiological significance of selective autophagy during animal development.
Asunto(s)
Autofagia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/embriología , Proteínas Portadoras/metabolismo , Gránulos Citoplasmáticos/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Trastornos del Desarrollo Sexual , Herencia Extracromosómica , Masculino , Datos de Secuencia Molecular , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Liquid-liquid phase separation (LLPS) has emerged as a crucial mechanism for cellular compartmentalization. One prominent example of this is the stress granule. Found in various types of cells, stress granule is a biomolecular condensate formed through phase separation. It comprises numerous RNA and RNA-binding proteins. Over the past decades, substantial knowledge has been gained about the composition and dynamics of stress granules. SGs can regulate various signaling pathways and have been associated with numerous human diseases, such as neurodegenerative diseases, cancer, and infectious diseases. The threat of viral infections continues to loom over society. Both DNA and RNA viruses depend on host cells for replication. Intriguingly, many stages of the viral life cycle are closely tied to RNA metabolism in human cells. The field of biomolecular condensates has rapidly advanced in recent times. In this context, we aim to summarize research on stress granules and their link to viral infections. Notably, stress granules triggered by viral infections behave differently from the canonical stress granules triggered by sodium arsenite (SA) and heat shock. Studying stress granules in the context of viral infections could offer a valuable platform to link viral replication processes and host anti-viral responses. A deeper understanding of these biological processes could pave the way for innovative interventions and treatments for viral infectious diseases. They could potentially bridge the gap between basic biological processes and interactions between viruses and their hosts.
Asunto(s)
Fenómenos Biológicos , Virosis , Humanos , Gránulos Citoplasmáticos/metabolismo , Gránulos de Estrés , ARN/metabolismo , Virosis/metabolismo , Replicación ViralRESUMEN
The selective degradation of intracellular components by autophagy involves sequential interactions of the cargo with a receptor, which also binds the autophagosomal protein Atg8 and a scaffold protein. Here, we demonstrated that mutations in C. elegans epg-11, which encodes an arginine methyltransferase homologous to PRMT1, cause the defective removal of PGL-1 and PGL-3 (cargo)-SEPA-1 (receptor) complexes, known as PGL granules, from somatic cells during embryogenesis. Autophagic degradation of the PGL granule scaffold protein EPG-2 and other protein aggregates was unaffected in epg-11/prmt-1 mutants. Loss of epg-11/prmt-1 activity impairs the association of PGL granules with EPG-2 and LGG-1 puncta. EPG-11/PRMT-1 directly methylates arginines in the RGG domains of PGL-1 and PGL-3. Autophagic removal of PGL proteins is impaired when the methylated arginines are mutated. Our study reveals that posttranslational arginine methylation regulates the association of the cargo-receptor complex with the scaffold protein, providing a mechanism for modulating degradation efficiency in selective autophagy.
Asunto(s)
Arginina/genética , Autofagia/genética , Proteínas de Caenorhabditis elegans/genética , Desarrollo Embrionario , Proteolisis , Proteínas de Unión al ARN/genética , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Embrión no Mamífero/metabolismo , Metilación , Mutación , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
RATIONALE: Growth differentiation factor 11 (GDF11) and GDF8 are members of the transforming growth factor-ß superfamily sharing 89% protein sequence homology. We have previously shown that circulating GDF11 levels decrease with age in mice. However, a recent study by Egerman et al reported that GDF11/8 levels increase with age in mouse serum. OBJECTIVE: Here, we clarify the direction of change of circulating GDF11/8 levels with age and investigate the effects of GDF11 administration on the murine heart. METHODS AND RESULTS: We validated our previous finding that circulating levels of GDF11/8 decline with age in mice, rats, horses, and sheep. Furthermore, we showed by Western analysis that the apparent age-dependent increase in GDF11 levels, as reported by Egerman et al, is attributable to cross-reactivity of the anti-GDF11 antibody with immunoglobulin, which is known to increase with age. GDF11 administration in mice rapidly activated SMAD2 and SMAD3 signaling in myocardium in vivo and decreased cardiac mass in both young (2-month-old) and old (22-month-old) mice in a dose-dependent manner after only 9 days. CONCLUSIONS: Our study confirms an age-dependent decline in serum GDF11/8 levels in multiple mammalian species and that exogenous GDF11 rapidly activates SMAD signaling and reduces cardiomyocyte size. Unraveling the molecular basis for the age-dependent decline in GDF11/8 could yield insight into age-dependent cardiac pathologies.
Asunto(s)
Envejecimiento/sangre , Proteínas Morfogenéticas Óseas/sangre , Factores de Diferenciación de Crecimiento/sangre , Miostatina/sangre , Animales , Biomarcadores/sangre , Caballos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas , OvinosRESUMEN
Liquid-liquid phase separation (LLPS) mediated by G3BP1/2 proteins and non-translating mRNAs mediates stress granule (SG) assembly. We investigated the phylogenetic evolution of G3BP orthologs from unicellular yeast to mammals and identified both conserved and divergent features. The modular domain organization of G3BP orthologs is generally conserved. However, invertebrate orthologs displayed reduced capacity for SG assembly in human cells compared to vertebrate orthologs. We demonstrated that the protein-interaction network facilitated by the NTF2L domain is a crucial determinant of this specificity. The evolution of the G3BP1 network coincided with its exploitation by certain viruses, as evident from the interaction between viral proteins and G3BP orthologs in insects and vertebrates. We revealed the importance and divergence of the G3BP interaction network in human SG formation. Leveraging this network, we established a 7-component in vitro SG reconstitution system for quantitative studies. These findings highlight the significance of G3BP network divergence in the evolution of biological processes.
Asunto(s)
ADN Helicasas , Proteínas de Unión a Poli-ADP-Ribosa , Mapas de Interacción de Proteínas , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Gránulos de Estrés , Humanos , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Helicasas/metabolismo , ARN Helicasas/genética , Gránulos de Estrés/metabolismo , Animales , ADN Helicasas/metabolismo , ADN Helicasas/genética , Filogenia , Células HeLa , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Proteínas de Unión al ARN , Proteínas Adaptadoras Transductoras de SeñalesRESUMEN
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
Liquid-liquid phase separation (LLPS) is a mechanism of intracellular organization that underlies the assembly of a variety of RNP granules. Fundamental biophysical principles governing LLPS during granule assembly have been revealed by simple in vitro systems, but these systems have limitations when studying the biology of complex, multicomponent RNP granules. Visualization of RNP granules in cells has validated key principles revealed by simple in vitro systems, but this approach presents difficulties for interrogating biophysical features of RNP granules and provides limited ability to manipulate protein, nucleic acid, or small molecule concentrations. Here, we introduce a system that builds upon recent insights into the mechanisms underlying RNP granule assembly and permits high-fidelity reconstitution of stress granules and the granular component of nucleoli in mammalian cellular lysate. This system fills the gap between simple in vitro systems and live cells and allows for a variety of studies of membraneless organelles, including the development of therapeutics that modify properties of specific condensates.
Asunto(s)
Nucléolo Celular/metabolismo , Gránulos Citoplasmáticos/metabolismo , Mamíferos/metabolismo , Estrés Fisiológico , Animales , Extractos Celulares , Línea Celular , ADN Helicasas/aislamiento & purificación , ADN Helicasas/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Nucleofosmina , Proteínas de Unión a Poli-ADP-Ribosa/aislamiento & purificación , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN/metabolismo , ARN Helicasas/aislamiento & purificación , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/aislamiento & purificación , Proteínas con Motivos de Reconocimiento de ARN/metabolismoRESUMEN
Prion-like proteins form multivalent assemblies and phase separate into membraneless organelles. Heterogeneous ribonucleoprotein D-like (hnRNPDL) is a RNA-processing prion-like protein with three alternative splicing (AS) isoforms, which lack none, one, or both of its two disordered domains. It has been suggested that AS might regulate the assembly properties of RNA-processing proteins by controlling the incorporation of multivalent disordered regions in the isoforms. This, in turn, would modulate their activity in the downstream splicing program. Here, we demonstrate that AS controls the phase separation of hnRNPDL, as well as the size and dynamics of its nuclear complexes, its nucleus-cytoplasm shuttling, and amyloidogenicity. Mutation of the highly conserved D378 in the disordered C-terminal prion-like domain of hnRNPDL causes limb-girdle muscular dystrophy 1G. We show that D378H/N disease mutations impact hnRNPDL assembly properties, accelerating aggregation and dramatically reducing the protein solubility in the muscle of Drosophila, suggesting a genetic loss-of-function mechanism for this muscular disorder.
Asunto(s)
Proteínas Amiloidogénicas/metabolismo , Núcleo Celular/metabolismo , Drosophila/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo D/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo D/metabolismo , Distrofia Muscular de Cinturas/genética , Agregación Patológica de Proteínas/metabolismo , Empalme Alternativo , Proteínas Amiloidogénicas/genética , Proteínas Amiloidogénicas/ultraestructura , Animales , Núcleo Celular/efectos de los fármacos , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Dactinomicina/farmacología , Drosophila/metabolismo , Técnicas de Inactivación de Genes , Células HeLa , Ribonucleoproteína Heterogénea-Nuclear Grupo D/ultraestructura , Humanos , Cinética , Microscopía Electrónica de Transmisión , Células Musculares/metabolismo , Células Musculares/patología , Distrofia Muscular de Cinturas/metabolismo , Mutación , Agregación Patológica de Proteínas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestructuraRESUMEN
Stress granules (SGs) are non-membrane-bound RNA-protein granules that assemble through phase separation in response to cellular stress. Disturbances in SG dynamics have been implicated as a primary driver of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), suggesting the hypothesis that these diseases reflect an underlying disturbance in the dynamics and material properties of SGs. However, this concept has remained largely untestable in available models of SG assembly, which require the confounding variable of exogenous stressors. Here we introduce a light-inducible SG system, termed OptoGranules, based on optogenetic multimerization of G3BP1, which is an essential scaffold protein for SG assembly. In this system, which permits experimental control of SGs in living cells in the absence of exogenous stressors, we demonstrate that persistent or repetitive assembly of SGs is cytotoxic and is accompanied by the evolution of SGs to cytoplasmic inclusions that recapitulate the pathology of ALS-FTD. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).
Asunto(s)
Esclerosis Amiotrófica Lateral/fisiopatología , Gránulos Citoplasmáticos/metabolismo , ADN Helicasas/metabolismo , Demencia Frontotemporal/fisiopatología , Modelos Teóricos , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Estrés Fisiológico , Línea Celular , Supervivencia Celular , Humanos , Optogenética/métodos , Estimulación LuminosaRESUMEN
Ribosome degradation through the autophagy-lysosome pathway is crucial for cell survival during nutrient starvation, but whether it occurs under normal growth conditions and contributes to animal physiology remains unaddressed. In this study, we identified RNST-2, a C. elegans T2 family endoribonuclease, as the key enzyme that degrades ribosomal RNA in lysosomes. We found that loss of rnst-2 causes accumulation of rRNA and ribosomal proteins in enlarged lysosomes and both phenotypes are suppressed by blocking autophagy, which indicates that RNST-2 mediates autophagic degradation of ribosomal RNA in lysosomes. rnst-2(lf) mutants are defective in embryonic and larval development and are short-lived. Remarkably, simultaneous loss of RNST-2 and de novo synthesis of pyrimidine nucleotides leads to complete embryonic lethality, which is suppressed by supplements of uridine or cytidine. Our study reveals an essential role of autophagy-dependent degradation of ribosomal RNA in maintaining nucleotide homeostasis during animal development.
Asunto(s)
Autofagia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Homeostasis , Nucleótidos/metabolismo , Estabilidad del ARN , ARN Ribosómico/metabolismo , Ribonucleasas/metabolismo , Animales , Caenorhabditis elegans/enzimología , Proteínas de Caenorhabditis elegans/genética , Endorribonucleasas , Eliminación de Gen , Lisosomas/enzimología , Lisosomas/metabolismo , Ribonucleasas/genéticaRESUMEN
Autophagy involves the sequestration of a portion of the cytosolic contents in an enclosed double-membrane autophagosomal structure and its subsequent delivery to lysosomes for degradation. Autophagy activity functions in multiple biological processes during Caenorhabditis elegans development. The basal level of autophagy in embryos removes aggregate-prone proteins, paternal mitochondria and spermatid-specific membranous organelles (MOs). Autophagy also contributes to the efficient removal of embryonic apoptotic cell corpses by promoting phagosome maturation. During larval development, autophagy modulates miRNA-mediated gene silencing by selectively degrading AIN-1, a component of miRNA-induced silencing complex, and thus participates in the specification of multiple cell fates controlled by miRNAs. During development of the hermaphrodite germline, autophagy acts coordinately with the core apoptotic machinery to execute genotoxic stress-induced germline cell death and also cell death when caspase activity is partially compromised. Autophagy is also involved in the utilization of lipid droplets in the aging process in adult animals. Studies in C. elegans provide valuable insights into the physiological functions of autophagy in the development of multicellular organisms.