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1.
Am J Hum Genet ; 105(3): 509-525, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31422817

RESUMO

The human RNA helicase DDX6 is an essential component of membrane-less organelles called processing bodies (PBs). PBs are involved in mRNA metabolic processes including translational repression via coordinated storage of mRNAs. Previous studies in human cell lines have implicated altered DDX6 in molecular and cellular dysfunction, but clinical consequences and pathogenesis in humans have yet to be described. Here, we report the identification of five rare de novo missense variants in DDX6 in probands presenting with intellectual disability, developmental delay, and similar dysmorphic features including telecanthus, epicanthus, arched eyebrows, and low-set ears. All five missense variants (p.His372Arg, p.Arg373Gln, p.Cys390Arg, p.Thr391Ile, and p.Thr391Pro) are located in two conserved motifs of the RecA-2 domain of DDX6 involved in RNA binding, helicase activity, and protein-partner binding. We use functional studies to demonstrate that the first variants identified (p.Arg373Gln and p.Cys390Arg) cause significant defects in PB assembly in primary fibroblast and model human cell lines. These variants' interactions with several protein partners were also disrupted in immunoprecipitation assays. Further investigation via complementation assays included the additional variants p.Thr391Ile and p.Thr391Pro, both of which, similarly to p.Arg373Gln and p.Cys390Arg, demonstrated significant defects in P-body assembly. Complementing these molecular findings, modeling of the variants on solved protein structures showed distinct spatial clustering near known protein binding regions. Collectively, our clinical and molecular data describe a neurodevelopmental syndrome associated with pathogenic missense variants in DDX6. Additionally, we suggest DDX6 join the DExD/H-box genes DDX3X and DHX30 in an emerging class of neurodevelopmental disorders involving RNA helicases.

3.
Mol Cell ; 68(1): 144-157.e5, 2017 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-28965817

RESUMO

Within cells, soluble RNPs can switch states to coassemble and condense into liquid or solid bodies. Although these phase transitions have been reconstituted in vitro, for endogenous bodies the diversity of the components, the specificity of the interaction networks, and the function of the coassemblies remain to be characterized. Here, by developing a fluorescence-activated particle sorting (FAPS) method to purify cytosolic processing bodies (P-bodies) from human epithelial cells, we identified hundreds of proteins and thousands of mRNAs that structure a dense network of interactions, separating P-body from non-P-body RNPs. mRNAs segregating into P-bodies are translationally repressed, but not decayed, and this repression explains part of the poor genome-wide correlation between RNA and protein abundance. P-bodies condense thousands of mRNAs that strikingly encode regulatory processes. Thus, we uncovered how P-bodies, by condensing and segregating repressed mRNAs, provide a physical substrate for the coordinated regulation of posttranscriptional mRNA regulons.


Assuntos
Regulação da Expressão Gênica , Proteoma/genética , RNA Mensageiro/genética , Regulon , Ribonucleoproteínas/genética , Fracionamento Celular , Citoplasma/metabolismo , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , Ontologia Genética , Células HEK293 , Células HeLa , Humanos , Anotação de Sequência Molecular , Transição de Fase , Biossíntese de Proteínas , Proteoma/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo
4.
Nucleic Acids Res ; 44(13): 6318-34, 2016 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-27342281

RESUMO

4E-Transporter binds eIF4E via its consensus sequence YXXXXLΦ, shared with eIF4G, and is a nucleocytoplasmic shuttling protein found enriched in P-(rocessing) bodies. 4E-T inhibits general protein synthesis by reducing available eIF4E levels. Recently, we showed that 4E-T bound to mRNA however represses its translation in an eIF4E-independent manner, and contributes to silencing of mRNAs targeted by miRNAs. Here, we address further the mechanism of translational repression by 4E-T by first identifying and delineating the interacting sites of its major partners by mass spectrometry and western blotting, including DDX6, UNR, unrip, PAT1B, LSM14A and CNOT4. Furthermore, we document novel binding between 4E-T partners including UNR-CNOT4 and unrip-LSM14A, altogether suggesting 4E-T nucleates a complex network of RNA-binding protein interactions. In functional assays, we demonstrate that joint deletion of two short conserved motifs that bind UNR and DDX6 relieves repression of 4E-T-bound mRNA, in part reliant on the 4E-T-DDX6-CNOT1 axis. We also show that the DDX6-4E-T interaction mediates miRNA-dependent translational repression and de novo P-body assembly, implying that translational repression and formation of new P-bodies are coupled processes. Altogether these findings considerably extend our understanding of the role of 4E-T in gene regulation, important in development and neurogenesis.


Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos/genética , Sítios de Ligação , RNA Helicases DEAD-box/genética , Proteínas de Ligação a DNA/genética , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4G em Eucariotos/genética , Fator de Iniciação 4G em Eucariotos/metabolismo , Regulação da Expressão Gênica/genética , Células HEK293 , Células HeLa , Humanos , Proteínas de Transporte Nucleocitoplasmático/genética , Ligação Proteica , Mapas de Interação de Proteínas/genética , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética
5.
Mol Biol Cell ; 26(14): 2579-95, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25995375

RESUMO

P-bodies are cytoplasmic ribonucleoprotein granules involved in posttranscriptional regulation. DDX6 is a key component of their assembly in human cells. This DEAD-box RNA helicase is known to be associated with various complexes, including the decapping complex, the CPEB repression complex, RISC, and the CCR4/NOT complex. To understand which DDX6 complexes are required for P-body assembly, we analyzed the DDX6 interactome using the tandem-affinity purification methodology coupled to mass spectrometry. Three complexes were prominent: the decapping complex, a CPEB-like complex, and an Ataxin2/Ataxin2L complex. The exon junction complex was also found, suggesting DDX6 binding to newly exported mRNAs. Finally, some DDX6 was associated with polysomes, as previously reported in yeast. Despite its high enrichment in P-bodies, most DDX6 is localized out of P-bodies. Of the three complexes, only the decapping and CPEB-like complexes were recruited into P-bodies. Investigation of P-body assembly in various conditions allowed us to distinguish required proteins from those that are dispensable or participate only in specific conditions. Three proteins were required in all tested conditions: DDX6, 4E-T, and LSM14A. These results reveal the variety of pathways of P-body assembly, which all nevertheless share three key factors connecting P-body assembly to repression.


Assuntos
Grânulos Citoplasmáticos/metabolismo , RNA Helicases DEAD-box/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ribonucleoproteínas/metabolismo , Ataxina-2/metabolismo , Humanos , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ligação Proteica , RNA Mensageiro/metabolismo
6.
RNA ; 18(9): 1702-15, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22836354

RESUMO

Translational repression is achieved by protein complexes that typically bind 3' UTR mRNA motifs and interfere with the formation of the cap-dependent initiation complex, resulting in mRNPs with a closed-loop conformation. We demonstrate here that the human DEAD-box protein Rck/p54, which is a component of such complexes and central to P-body assembly, is in considerable molecular excess with respect to cellular mRNAs and enriched to a concentration of 0.5 mM in P-bodies, where it is organized in clusters. Accordingly, multiple binding of p54 proteins along mRNA molecules was detected in vivo. Consistently, the purified protein bound RNA with no sequence specificity and high nanomolar affinity. Moreover, bound RNA molecules had a relaxed conformation. While RNA binding was ATP independent, relaxing of bound RNA was dependent on ATP, though not on its hydrolysis. We propose that Rck/p54 recruitment by sequence-specific translational repressors leads to further binding of Rck/p54 along mRNA molecules, resulting in their masking, unwinding, and ultimately recruitment to P-bodies. Rck/p54 proteins located at the 5' extremity of mRNA can then recruit the decapping complex, thus coupling translational repression and mRNA degradation.


Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/metabolismo , Trifosfato de Adenosina/metabolismo , Células HeLa , Humanos , Modelos Biológicos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica
7.
Biochimie ; 94(7): 1572-7, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22445682

RESUMO

Micro-RNAs (miRNAs) are major actors of RNA interference (RNAi), a regulation pathway which leads to translational repression and/or degradation of specific mRNAs. They provide target specificity by incorporating into the RISC complex and guiding its binding to mRNA. Since the discovery of RNAi, many progresses have been made on the mechanism of action of the RISC complex and on the identification of target mRNAs. However, the regulation of RNAi has been poorly investigated so far. Recently, various studies have revealed physical and functional relationships between RNAi, P-bodies and mitochondria. This review intends to recapitulate these data and discuss their potential importance in cell metabolism.


Assuntos
Grânulos Citoplasmáticos/genética , Mitocôndrias/genética , Interferência de RNA , Animais , Linhagem Celular , Citosol/metabolismo , Humanos , MicroRNAs/genética
9.
J Biol Chem ; 286(27): 24219-30, 2011 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-21576251

RESUMO

P-bodies are cytoplasmic granules that are linked to mRNA decay, mRNA storage, and RNA interference (RNAi). They are known to interact with stress granules in stressed cells, and with late endosomes. Here, we report that P-bodies also interact with mitochondria, as previously described for P-body-related granules in germ cells. The interaction is dynamic, as a large majority of P-bodies contacts mitochondria at least once within a 3-min interval, and for about 18 s. This association requires an intact microtubule network. The depletion of P-bodies does not seem to affect mitochondria, nor the mitochondrial activity to be required for their contacts with P-bodies. However, inactivation of mitochondria leads to a strong decrease of miRNA-mediated RNAi efficiency, and to a lesser extent of siRNA-mediated RNAi. The defect occurs during the assembly of active RISC and is associated with a specific delocalization of endogeneous Ago2 from P-bodies. Our study reveals the possible involvement of RNAi defect in pathologies involving mitochondrial deficiencies.


Assuntos
Grânulos Citoplasmáticos/metabolismo , MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Interferência de RNA , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas Argonauta , Grânulos Citoplasmáticos/genética , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Células HEK293 , Células HeLa , Humanos , MicroRNAs/genética , Mitocôndrias/genética , Mitocôndrias/patologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Transporte Proteico/genética , RNA Mensageiro/genética
10.
FEBS J ; 276(11): 3137-47, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19490114

RESUMO

Human MOK2 is a DNA-binding transcriptional repressor. Previously, we identified nuclear lamin A/C proteins as protein partners of hsMOK2. Furthermore, we found that a fraction of hsMOK2 protein was associated with the nuclear matrix. We therefore suggested that hsMOK2 interactions with lamin A/C and the nuclear matrix may be important for its ability to repress transcription. In this study, we identify JNK-associated leucine zipper and JSAP1 scaffold proteins, two members of c-Jun N-terminal kinase (JNK)-interacting proteins family as partners of hsMOK2. Because these results suggested that hsMOK2 could be phosphorylated, we investigated the phosphorylation status of hsMOK2. We identified Ser38 and Ser129 of hsMOK2 as phosphorylation sites of JNK3 kinase, and Ser46 as a phosphorylation site of Aurora A and protein kinase A. These three serine residues are located in the lamin A/C-binding domain. Interestingly, we were able to demonstrate that the phosphorylation of hsMOK2 interfered with its ability to bind lamin A/C.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Lamina Tipo A/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Aurora Quinases , Sítios de Ligação , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Poliacrilamida , Ensaio de Desvio de Mobilidade Eletroforética , Células HeLa , Humanos , Lamina Tipo A/genética , Microscopia de Fluorescência , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Serina/genética , Serina/metabolismo , Dedos de Zinco
11.
Mol Biol Cell ; 20(1): 176-87, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18923137

RESUMO

The translational regulator CPEB1 plays a major role in the control of maternal mRNA in oocytes, as well as of subsynaptic mRNAs in neurons. Although mainly cytoplasmic, we found that CPEB1 protein is continuously shuttling between nucleus and cytoplasm. Its export is controlled by two redundant NES motifs dependent on the nuclear export receptor Crm1. In the nucleus, CPEB1 accumulates in a few foci most often associated with nucleoli. These foci are different from previously identified nuclear bodies. They contain Crm1 and were called Crm1 nucleolar bodies (CNoBs). CNoBs depend on RNA polymerase I activity, indicating a role in ribosome biogenesis. However, although they form in the nucleolus, they never migrate to the nuclear envelope, precluding a role as a mediator for ribosome export. They could rather constitute a platform providing factors for ribosome assembly or export. The behavior of CPEB1 in CNoBs raises the possibility that it is involved in ribosome biogenesis.


Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Nucléolo Celular/metabolismo , Carioferinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Motivos de Aminoácidos , Animais , Nucléolo Celular/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Carioferinas/genética , Sinais de Exportação Nuclear , Biossíntese de Proteínas , RNA Polimerase I/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética
12.
Biol Cell ; 100(1): 51-61, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17760566

RESUMO

BACKGROUND INFORMATION: hsMOK2 (human MOK2) is a DNA-binding transcriptional repressor. For example, it represses the IRBP (interphotoreceptor retinoid-binding protein) gene by competing with the CRX (cone-rod homeobox protein) transcriptional activator for DNA binding. Previous studies have shown an interaction between hsMOK2 and nuclear lamin A/C. This interaction could be important to explain hsMOK2 ability to repress transcription. RESULTS: In the present study, we have tested whether missense pathogenic mutations of lamin A/C, which are located in the hsMOK2-binding domain, could affect the interaction with hsMOK2. We find that none of the tested mutations is able to disrupt hsMOK2 binding in vitro or in vivo. However, we observe an aberrant cellular localization of hsMOK2 into nuclear aggregates when pathogenic lamin A/C mutant proteins are expressed. CONCLUSIONS: These results indicate that pathogenic mutations in lamin A/C lead to sequestration of hsMOK2 into nuclear aggregates, which may deregulate MOK2 target genes.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Lamina Tipo A , Mutação de Sentido Incorreto , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
13.
Nucleic Acids Res ; 30(21): 4634-42, 2002 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-12409453

RESUMO

The human and murine MOK2 proteins are factors able to recognize both DNA and RNA through their zinc finger motifs. This dual affinity of MOK2 suggests that MOK2 might be involved in transcription and post-transcriptional regulation of MOK2 target genes. The IRBP gene contains two MOK2-binding elements, a complete 18 bp MOK2-binding site located in intron 2 and the essential core MOK2-binding site (8 bp of conserved 3'-half-site) located in the IRBP promoter. We have demonstrated that MOK2 can bind to the 8 bp present in the IRBP promoter and repress transcription from this promoter by competing with the CRX activator for DNA binding. In this study, we identify a novel interaction between lamin A/C and hsMOK2 by using the yeast two-hybrid system. The interaction, which was confirmed by GST pull-down assays and co-immunolocalization studies in vivo, requires the N-terminal acidic domain of hsMOK2 and the coiled 2 domain of lamin A/C. Furthermore, we show that a fraction of hsMOK2 protein is associated with the nuclear matrix. We therefore suggest that hsMOK2 interactions with lamin A/C and the nuclear matrix may be important for its ability to repress transcription.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Lamina Tipo A/metabolismo , Fatores de Transcrição/metabolismo , Sítios de Ligação , Núcleo Celular/química , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Lamina Tipo A/química , Lamina Tipo A/genética , Matriz Nuclear/química , Matriz Nuclear/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Fatores de Transcrição/química , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido
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