Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 13 de 13
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nucleus ; 3(4): 320-1, 2012 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-22688646

RESUMO

It is widely accepted that protein synthesis occurs in the cytoplasms of eukaryotic cells, but some investigators believe that it also occurs in the nucleus. In spite of experiments performed in several labs over many years, the issue of nuclear translation remains unresolved. Advocates assert that it would serve as an economical and convenient way to explain how cells monitor the quality of newly made mRNAs or ribosomes. Skeptics argue that regardless of its esthetic appeal, compelling evidence for nuclear translation has been absent. The key question--also central to the debate more than 30 years ago--is whether alleged nuclear translation can be proven to represent "genuine polypeptide synthesis that is a function of the nuclear compartment".


Assuntos
Núcleo Celular/metabolismo , Peptidil Transferases/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Peptidil Transferases/antagonistas & inibidores , Puromicina/química , Puromicina/farmacologia , Relação Estrutura-Atividade
2.
Genes Dev ; 25(11): 1121-31, 2011 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21576259

RESUMO

We show that, in Xenopus laevis oocytes and early embryos, double-stranded exogenous siRNAs cannot function as microRNA (miRNA) mimics in either deadenylation or guided mRNA cleavage (RNAi). Instead, siRNAs saturate and inactivate maternal Argonaute (Ago) proteins, which are present in low amounts but are needed for Dicer processing of pre-miRNAs at the midblastula transition (MBT). Consequently, siRNAs impair accumulation of newly made miRNAs, such as the abundant embryonic pre-miR-427, but inhibition dissipates upon synthesis of zygotic Ago proteins after MBT. These effects of siRNAs, which are independent of sequence, result in morphological defects at later stages of development. The expression of any of several exogenous human Ago proteins, including catalytically inactive Ago2 (Ago2mut), can overcome the siRNA-mediated inhibition of miR-427 biogenesis and function. However, expression of wild-type, catalytically active hAgo2 is required to elicit RNAi in both early embryos and oocytes using either siRNA or endogenous miRNAs as guides. The lack of endogenous Ago2 endonuclease activity explains why these cells normally are unable to support RNAi. Expression of catalytically active exogenous Ago2, which appears not to perturb normal Xenopus embryonic development, can now be exploited for RNAi in this vertebrate model organism.


Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/biossíntese , Interferência de RNA , Xenopus laevis/embriologia , Animais , Proteínas Argonautas , Embrião não Mamífero , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Oócitos/metabolismo , Estabilidade de RNA , RNA Interferente Pequeno , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/metabolismo , Xenopus laevis/genética
3.
PLoS Biol ; 8(10): e1000522, 2010 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-21048991

RESUMO

The assembly of ribosomal subunits in eukaryotes is a complex, multistep process so far mostly studied in yeast. In S. cerevisiae, more than 200 factors including ribosomal proteins and trans-acting factors are required for the ordered assembly of 40S and 60S ribosomal subunits. To date, only few human homologs of these yeast ribosome synthesis factors have been characterized. Here, we used a systematic RNA interference (RNAi) approach to analyze the contribution of 464 candidate factors to ribosomal subunit biogenesis in human cells. The screen was based on visual readouts, using inducible, fluorescent ribosomal proteins as reporters. By performing computer-based image analysis utilizing supervised machine-learning techniques, we obtained evidence for a functional link of 153 human proteins to ribosome synthesis. Our data show that core features of ribosome assembly are conserved from yeast to human, but differences exist for instance with respect to 60S subunit export. Unexpectedly, our RNAi screen uncovered a requirement for the export receptor Exportin 5 (Exp5) in nuclear export of 60S subunits in human cells. We show that Exp5, like the known 60S exportin Crm1, binds to pre-60S particles in a RanGTP-dependent manner. Interference with either Exp5 or Crm1 function blocks 60S export in both human cells and frog oocytes, whereas 40S export is compromised only upon inhibition of Crm1. Thus, 60S subunit export is dependent on at least two RanGTP-binding exportins in vertebrate cells.


Assuntos
Carioferinas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Ribossomos/metabolismo , Animais , Células HeLa , Humanos , Carioferinas/genética , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/genética , Ribossomos/genética , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismo
4.
RNA ; 15(12): 2351-63, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19854872

RESUMO

We show that microRNA-427 (miR-427) mediates the rapid deadenylation of maternal mRNAs after the midblastula transition (MBT) of Xenopus laevis embryogenesis. By MBT, the stage when the embryonic cell cycle is remodeled and zygotic transcription of mRNAs is initiated, each embryo has accumulated approximately 10(9) molecules of miR-427 processed from multimeric pri-miR-427 transcripts synthesized after fertilization. We demonstrate that the maternal mRNAs for cyclins A1 and B2 each contain a single miR-427 target sequence, spanning less than 30 nucleotides, that is both necessary and sufficient for deadenylation, and that inactivation of miR-427 leads to stabilization of the mRNAs. Although this deadenylation normally takes place after MBT, exogenous miRNAs produced prematurely in vivo can promote deadenylation prior to MBT, indicating that turnover of the maternal mRNAs is limited by the amount of accumulated miR-427. Injected transcripts comprised solely of the cyclin mRNA 3' untranslated regions or bearing a 5' ApppG cap undergo deadenylation, showing that translation of the targeted RNA is not required. miR-427 is not unique in promoting deadenylation, as an unrelated miRNA, let-7, can substitute for miR-427 if the reporter RNA contains an appropriate let-7 target site. We propose that miR-427, like the orthologous miR-430 of zebrafish, functions to down-regulate expression of maternal mRNAs early in development.


Assuntos
Adenina/metabolismo , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Xenopus laevis/metabolismo , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Ciclina B2/genética , Ciclina B2/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Dados de Sequência Molecular , Biossíntese de Proteínas , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genética
5.
Sci STKE ; 2007(387): pe25, 2007 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-17519424

RESUMO

The innate immune response can be initiated by the binding of various pathogen-associated compounds or cytokines to receptors on the surfaces of dendritic cells. These interactions result in the activation of many genes and gene products. Several different pathways converge to raise the abundance of specific microRNAs (miRNAs). In particular, activation of the transcription factors AP-1 and NF-kappaB results in an increase in the amount of miR-155. High levels of this miRNA are associated with several types of cancer. However, the mRNAs that may be targeted by miR-155 in the innate immune response remain to be determined.


Assuntos
Imunidade Inata/genética , Imunidade Inata/imunologia , MicroRNAs/metabolismo , Animais , Galinhas , Citocinas/imunologia , Humanos , Camundongos , MicroRNAs/genética , Neoplasias/genética , RNA Neoplásico/metabolismo , Receptores Toll-Like/metabolismo
6.
Mol Cell ; 19(3): 292-4, 2005 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-16061175

RESUMO

Two recently published papers (Takano et al., 2005 and Shaheen and Hopper, 2005) demonstrate that in S. cerevisiae, cytoplasmic tRNAs can be transported into the nucleus. This retrograde movement may expose mature tRNAs to nuclear proofreading or it may regulate tRNA availability in response to amino acid availability.


Assuntos
Núcleo Celular/metabolismo , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Aminoácidos/deficiência , Aminoácidos/metabolismo , Citoplasma/metabolismo , Modelos Biológicos , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Splicing de RNA , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA de Transferência/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína ran de Ligação ao GTP/metabolismo
7.
Proc Natl Acad Sci U S A ; 102(10): 3627-32, 2005 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-15738415

RESUMO

We show that the microRNA miR-155 can be processed from sequences present in BIC RNA, a spliced and polyadenylated but non-protein-coding RNA that accumulates in lymphoma cells. The precursor of miR-155 is likely a transient spliced or unspliced nuclear BIC transcript rather than accumulated BIC RNA, which is primarily cytoplasmic. By using a sensitive and quantitative assay, we find that clinical isolates of several types of B cell lymphomas, including diffuse large B cell lymphoma (DLBCL), have 10- to 30-fold higher copy numbers of miR-155 than do normal circulating B cells. Similarly, the quantities of BIC RNA are elevated in lymphoma cells, but ratios of the amounts of the two RNAs are not constant, suggesting that the level of miR-155 is controlled by transcription and processing. Significantly higher levels of miR-155 are present in DLBCLs with an activated B cell phenotype than with the germinal center phenotype. Because patients with activated B cell-type DLBCL have a poorer clinical prognosis, quantification of this microRNA may be diagnostically useful.


Assuntos
Linfoma de Células B/genética , Linfoma Difuso de Grandes Células B/genética , MicroRNAs/metabolismo , RNA não Traduzido/metabolismo , Linhagem Celular Tumoral , Humanos , MicroRNAs/análise , RNA Mensageiro/metabolismo , RNA não Traduzido/análise
8.
RNA ; 10(7): 1153-61, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15208450

RESUMO

The short lengths of microRNAs (miRNAs) present a significant challenge for detection and quantitation using conventional methods for RNA analysis. To address this problem, we developed a quantitative, sensitive, and rapid miRNA assay based on our previously described messenger RNA Invader assay. This assay was used successfully in the analysis of several miRNAs, using as little as 50-100 ng of total cellular RNA or as few as 1,000 lysed cells. Its specificity allowed for discrimination between miRNAs differing by a single nucleotide, and between precursor and mature miRNAs. The Invader miRNA assay, which can be performed in unfractionated detergent lysates, uses fluorescence detection in microtiter plates and requires only 2-3 h incubation time, allowing for parallel analysis of multiple samples in high-throughput screening analyses.


Assuntos
MicroRNAs/análise , MicroRNAs/genética , Sequência de Bases , Linhagem Celular , Técnicas Genéticas , Células HeLa , Humanos , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/química
9.
Curr Opin Cell Biol ; 16(3): 335-8, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15145360

RESUMO

Although it is universally accepted that protein synthesis occurs in the cytoplasm, the possibility that translation can also take place in the nucleus has been hotly debated. Reports have been published claiming to demonstrate nuclear translation, but alternative explanations for these results have not been excluded, and other experiments argue against it. Much of the appeal of nuclear translation is that functional proofreading of newly made mRNAs in the nucleus would provide an efficient way to monitor mRNAs for the presence of premature termination codons, thereby avoiding the synthesis of deleterious proteins. mRNAs that are still in the nucleus-associated fraction of cells are subject to translational proofreading resulting in nonsense-mediated mRNA decay and perhaps nonsense-associated alternate splicing. However, these mRNAs are likely to be in the perinuclear cytoplasm rather than within the nucleus. Therefore, in the absence of additional evidence, we conclude that nuclear translation is unlikely to occur.


Assuntos
Núcleo Celular/metabolismo , Biossíntese de Proteínas/fisiologia , Animais , Núcleo Celular/genética , Códon de Terminação , Humanos , Precursores de RNA/biossíntese , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética
10.
Science ; 303(5654): 95-8, 2004 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-14631048

RESUMO

MicroRNAs (miRNAs), which function as regulators of gene expression in eukaryotes, are processed from larger transcripts by sequential action of nuclear and cytoplasmic ribonuclease III-like endonucleases. We show that Exportin-5 (Exp5) mediates efficient nuclear export of short miRNA precursors (pre-miRNAs) and that its depletion by RNA interference results in reduced miRNA levels. Exp5 binds correctly processed pre-miRNAs directly and specifically, in a Ran guanosine triphosphate-dependent manner, but interacts only weakly with extended pre-miRNAs that yield incorrect miRNAs when processed by Dicer in vitro. Thus, Exp5 is key to miRNA biogenesis and may help coordinate nuclear and cytoplasmic processing steps.


Assuntos
Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Carioferinas/metabolismo , MicroRNAs/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Precursores de RNA/metabolismo , Receptores Citoplasmáticos e Nucleares , Animais , Proteínas de Transporte/metabolismo , Citoplasma/metabolismo , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , MicroRNAs/química , Oócitos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/metabolismo , Ribonuclease III/metabolismo , Xenopus , Proteína ran de Ligação ao GTP/metabolismo , Proteína Exportina 1
11.
EMBO J ; 22(11): 2841-51, 2003 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-12773398

RESUMO

60S and 40S ribosomal subunits are assembled in the nucleolus and exported from the nucleus to the cytoplasm independently of each other. We show that in vertebrate cells, transport of both subunits requires the export receptor CRM1 and Ran.GTP. Export of 60S subunits is coupled with that of the nucleo- cytoplasmic shuttling protein NMD3. Human NMD3 (hNMD3) contains a CRM-1-dependent leucine-rich nuclear export signal (NES) and a complex, dispersed nuclear localization signal (NLS), the basic region of which is also required for nucleolar accumulation. When present in Xenopus oocytes, both wild-type and export-defective mutant hNMD3 proteins bind to newly made nuclear 60S pre-export particles at a late step of subunit maturation. The export-defective hNMD3, but not the wild-type protein, inhibits export of 60S subunits from oocyte nuclei. These results indicate that the NES mutant protein competes with endogenous wild-type frog NMD3 for binding to nascent 60S subunits, thereby preventing their export. We propose that NMD3 acts as an adaptor for CRM1-Ran.GTP-mediated 60S subunit export, by a mechanism that is conserved from vertebrates to yeast.


Assuntos
Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares , Ribossomos/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Ligação Competitiva , Sequência Conservada , Feminino , Células HeLa , Humanos , Técnicas In Vitro , Carioferinas/metabolismo , Dados de Sequência Molecular , Mutação , Sinais de Localização Nuclear/genética , Oócitos/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribossomos/química , Homologia de Sequência de Aminoácidos , Transfecção , Xenopus laevis , Proteína ran de Ligação ao GTP/metabolismo , Proteína Exportina 1
12.
RNA ; 9(1): 1-8, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12554869

RESUMO

Recently, several reports have been published in support of the idea that protein synthesis occurs in both the nucleus and the cytoplasm. This proposal has generated a great deal of excitement because, if true, it would mean that our thinking about the compartmentalization of cell functions would have to be re-evaluated. The significance and broad implications of this phenomenon require that the experimental evidence used to support it be carefully evaluated. Here, we critique the published evidence in support of, or in opposition to, the question of whether translation occurs in the nucleus. Arguments in support of nuclear translation focus on three issues: (1) the presence of translation factors and ribosomal components in the nucleus, and their recruitment to sites of transcription; (2) amino acid incorporation in isolated nuclei and in nuclei under conditions that should not permit protein import; and (3) the fact that nuclear translation would account for observations that are otherwise difficult to explain. Arguments against nuclear translation emphasize the absence (or low abundance) from nuclei of many translation factors; the likely inactivity of nascent ribosomes; and the loss of translation activity as nuclei are purified from contaminating cytoplasm. In our opinion, all of the experiments on nuclear translation published to date lack critical controls and, therefore, are not compelling; also, traditional mechanisms can explain the observations for which nuclear translation has been invoked. Thus, while we cannot rule out nuclear translation, in the absence of better supporting data we are reluctant to believe it occurs.


Assuntos
Núcleo Celular/genética , Biossíntese de Proteínas , Expressão Gênica
13.
Trends Biochem Sci ; 27(11): 580-5, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12417134

RESUMO

The partitioning of cells by a nuclear envelope ensures that precursors of ribosomes do not interact prematurely with other components of the translation machinery. Ribosomal subunits are assembled in nucleoli and exported to the cytoplasm in a CRM1/Ran-GTP-dependent fashion. Export of the large (60S) subunit requires a shuttling adaptor protein, NMD3, which binds to mature, correctly folded subunits. Immature or defective particles do not bind NMD3 and thus are excluded from the export pathway. This structural proofreading is extended into the cytoplasm, where it is believed that several energy-requiring steps release shuttling factors from the subunit, allowing it to function in translation.


Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Núcleo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Ligação a RNA , Receptores Citoplasmáticos e Nucleares , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae , Sequência Conservada , Proteínas Fúngicas/genética , Carioferinas/metabolismo , Mutação , Estrutura Terciária de Proteína , Subunidades Proteicas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Proteína Exportina 1
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...