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1.
Cell ; 186(4): 803-820.e25, 2023 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-36738734

RESUMO

Complex diseases often involve the interplay between genetic and environmental factors. Charcot-Marie-Tooth type 2 neuropathies (CMT2) are a group of genetically heterogeneous disorders, in which similar peripheral neuropathology is inexplicably caused by various mutated genes. Their possible molecular links remain elusive. Here, we found that upon environmental stress, many CMT2-causing mutant proteins adopt similar properties by entering stress granules (SGs), where they aberrantly interact with G3BP and integrate into SG pathways. For example, glycyl-tRNA synthetase (GlyRS) is translocated from the cytoplasm into SGs upon stress, where the mutant GlyRS perturbs the G3BP-centric SG network by aberrantly binding to G3BP. This disrupts SG-mediated stress responses, leading to increased stress vulnerability in motoneurons. Disrupting this aberrant interaction rescues SG abnormalities and alleviates motor deficits in CMT2D mice. These findings reveal a stress-dependent molecular link across diverse CMT2 mutants and provide a conceptual framework for understanding genetic heterogeneity in light of environmental stress.


Assuntos
Doença de Charcot-Marie-Tooth , Proteínas com Motivo de Reconhecimento de RNA , Grânulos de Estresse , Animais , Camundongos , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Citoplasma , Neurônios Motores , Proteínas com Motivo de Reconhecimento de RNA/metabolismo
2.
Cell ; 181(2): 325-345.e28, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32302571

RESUMO

The mechanisms underlying ribonucleoprotein (RNP) granule assembly, including the basis for establishing and maintaining RNP granules with distinct composition, are unknown. One prominent type of RNP granule is the stress granule (SG), a dynamic and reversible cytoplasmic assembly formed in eukaryotic cells in response to stress. Here, we show that SGs assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein-RNA interaction network. The central node of this network is G3BP1, which functions as a molecular switch that triggers RNA-dependent LLPS in response to a rise in intracellular free RNA concentrations. Moreover, we show that interplay between three distinct intrinsically disordered regions (IDRs) in G3BP1 regulates its intrinsic propensity for LLPS, and this is fine-tuned by phosphorylation within the IDRs. Further regulation of SG assembly arises through positive or negative cooperativity by extrinsic G3BP1-binding factors that strengthen or weaken, respectively, the core SG network.


Assuntos
Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Ribonucleoproteínas/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Estruturas Citoplasmáticas/metabolismo , Células HEK293 , Humanos , Fosforilação , RNA/metabolismo
3.
Cell ; 173(4): 958-971.e17, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29628143

RESUMO

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.


Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Esclerose Lateral Amiotrófica/patologia , Ataxina-2/metabolismo , Proteína C9orf72/genética , Demência Frontotemporal/patologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Idoso , Esclerose Lateral Amiotrófica/metabolismo , Arsenitos/toxicidade , Ataxina-2/antagonistas & inibidores , Ataxina-2/genética , Proteína C9orf72/metabolismo , Expansão das Repetições de DNA/genética , Feminino , Demência Frontotemporal/metabolismo , Células HEK293 , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Compostos de Sódio/toxicidade , alfa Carioferinas/antagonistas & inibidores , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , beta Carioferinas/antagonistas & inibidores , beta Carioferinas/genética , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/antagonistas & inibidores , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismo
4.
Mol Cell ; 84(9): 1633-1634, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38701739

RESUMO

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.


Assuntos
Fator de Iniciação Eucariótico 4G , Resposta ao Choque Térmico , Biossíntese de Proteínas , RNA Mensageiro , Fator de Iniciação Eucariótico 4G/metabolismo , Fator de Iniciação Eucariótico 4G/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Humanos , Animais , Conformação Proteica , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética
5.
Mol Cell ; 84(17): 3169-3171, 2024 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-39241750

RESUMO

In this issue of Molecular Cell, Xie et al.1 revealed that the proteasome is a constitutive component of plant stress granules (SGs), and that enhanced proteolytic activity is essential for efficient SG disassembly and plant survival during the stress response.


Assuntos
Grânulos Citoplasmáticos , Homeostase , Complexo de Endopeptidases do Proteassoma , Estresse Fisiológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Grânulos Citoplasmáticos/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteólise
6.
Mol Cell ; 79(4): 645-659.e9, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32692974

RESUMO

Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.


Assuntos
Grânulos Citoplasmáticos/genética , DNA Helicases/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Biossíntese de Proteínas , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Estresse Fisiológico/genética , Regiões 5' não Traduzidas , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/patologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Feminino , Células HCT116 , Células HeLa , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Espermatogônias/citologia , Espermatogônias/patologia , Testículo/citologia , Testículo/metabolismo
7.
Proc Natl Acad Sci U S A ; 121(23): e2322359121, 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38805286

RESUMO

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.


Assuntos
Proteína Adaptadora GRB2 , Sistema de Sinalização das MAP Quinases , Proteínas de Fusão Oncogênica , Proteínas Proto-Oncogênicas c-ret , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Proteínas ras , Humanos , Proteína Adaptadora GRB2/metabolismo , Proteína Adaptadora GRB2/genética , Células HEK293 , Proteínas de Fusão Oncogênica/metabolismo , Proteínas de Fusão Oncogênica/genética , Fosforilação , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas ras/metabolismo , Proteínas ras/genética , Transdução de Sinais , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/genética
8.
Cell ; 141(6): 1042-55, 2010 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-20550938

RESUMO

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.


Assuntos
Autofagia , Caenorhabditis elegans/genética , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Grânulos Citoplasmáticos/metabolismo , Lisossomos/metabolismo , Mutação , Fagossomos/metabolismo
9.
Cell ; 136(2): 308-21, 2009 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-19167332

RESUMO

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.


Assuntos
Autofagia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Proteínas de Transporte/metabolismo , Grânulos Citoplasmáticos/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Transtornos do Desenvolvimento Sexual , Herança Extracromossômica , Masculino , Dados de Sequência Molecular , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
10.
Acta Biochim Biophys Sin (Shanghai) ; 55(7): 1099-1118, 2023 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-37401177

RESUMO

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.


Assuntos
Fenômenos Biológicos , Viroses , Humanos , Grânulos Citoplasmáticos/metabolismo , Grânulos de Estresse , RNA/metabolismo , Viroses/metabolismo , Replicação Viral
11.
Mol Cell ; 52(3): 421-33, 2013 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-24140420

RESUMO

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.


Assuntos
Arginina/genética , Autofagia/genética , Proteínas de Caenorhabditis elegans/genética , Desenvolvimento Embrionário , Proteólise , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Embrião não Mamífero/metabolismo , Metilação , Mutação , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas de Ligação a RNA/metabolismo
12.
Nat Chem Biol ; 19(11): 1299-1300, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37592156
13.
Nat Chem Biol ; 19(10): 1180-1182, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37731018
14.
Circ Res ; 118(1): 29-37, 2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26489925

RESUMO

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.


Assuntos
Envelhecimento/sangue , Proteínas Morfogenéticas Ósseas/sangue , Fatores de Diferenciação de Crescimento/sangue , Miostatina/sangue , Animais , Biomarcadores/sangue , Cavalos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Ovinos
15.
Cell Rep ; 43(8): 114617, 2024 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-39120973

RESUMO

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.


Assuntos
DNA Helicases , Proteínas de Ligação a Poli-ADP-Ribose , Mapas de Interação de Proteínas , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Grânulos de Estresse , Humanos , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/metabolismo , RNA Helicases/genética , Grânulos de Estresse/metabolismo , Animais , DNA Helicases/metabolismo , DNA Helicases/genética , Filogenia , Células HeLa , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Proteínas de Ligação a RNA , Proteínas Adaptadoras de Transdução de Sinal
16.
Nat Commun ; 15(1): 4127, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38750080

RESUMO

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.


Assuntos
Infecções por Vírus de RNA , Grânulos de Estresse , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Humanos , Grânulos Citoplasmáticos/metabolismo , Proteína DEAD-box 58/metabolismo , DNA Helicases/metabolismo , Células HEK293 , Células HeLa , Imunidade Inata , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Receptores Imunológicos/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Infecções por Vírus de RNA/virologia , Infecções por Vírus de RNA/metabolismo , Infecções por Vírus de RNA/imunologia , RNA de Cadeia Dupla/metabolismo , Transdução de Sinais , Grânulos de Estresse/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
17.
J Cell Biol ; 220(3)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33502444

RESUMO

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.


Assuntos
Nucléolo Celular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Mamíferos/metabolismo , Estresse Fisiológico , Animais , Extratos Celulares , Linhagem Celular , DNA Helicases/isolamento & purificação , DNA Helicases/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Nucleofosmina , Proteínas de Ligação a Poli-ADP-Ribose/isolamento & purificação , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA/metabolismo , RNA Helicases/isolamento & purificação , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/isolamento & purificação , Proteínas com Motivo de Reconhecimento de RNA/metabolismo
18.
Cell Rep ; 30(4): 1117-1128.e5, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31995753

RESUMO

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.


Assuntos
Proteínas Amiloidogênicas/metabolismo , Núcleo Celular/metabolismo , Drosophila/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Distrofia Muscular do Cíngulo dos Membros/genética , Agregação Patológica de Proteínas/metabolismo , Processamento Alternativo , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/ultraestrutura , Animais , Núcleo Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Dactinomicina/farmacologia , Drosophila/metabolismo , Técnicas de Inativação de Genes , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/ultraestrutura , Humanos , Cinética , Microscopia Eletrônica de Transmissão , Células Musculares/metabolismo , Células Musculares/patologia , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Mutação , Agregação Patológica de Proteínas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestrutura
19.
Elife ; 82019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30893049

RESUMO

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).


Assuntos
Esclerose Lateral Amiotrófica/fisiopatologia , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Demência Frontotemporal/fisiopatologia , Modelos Teóricos , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Estresse Fisiológico , Linhagem Celular , Sobrevivência Celular , Humanos , Optogenética/métodos , Estimulação Luminosa
20.
Elife ; 72018 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-30102152

RESUMO

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.


Assuntos
Autofagia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Homeostase , Nucleotídeos/metabolismo , Estabilidade de RNA , RNA Ribossômico/metabolismo , Ribonucleases/metabolismo , Animais , Caenorhabditis elegans/enzimologia , Proteínas de Caenorhabditis elegans/genética , Endorribonucleases , Deleção de Genes , Lisossomos/enzimologia , Lisossomos/metabolismo , Ribonucleases/genética
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