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1.
Cell ; 157(3): 651-63, 2014 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-24766810

RESUMO

Neurodegenerative diseases can occur so early as to affect neurodevelopment. From a cohort of more than 2,000 consanguineous families with childhood neurological disease, we identified a founder mutation in four independent pedigrees in cleavage and polyadenylation factor I subunit 1 (CLP1). CLP1 is a multifunctional kinase implicated in tRNA, mRNA, and siRNA maturation. Kinase activity of the CLP1 mutant protein was defective, and the tRNA endonuclease complex (TSEN) was destabilized, resulting in impaired pre-tRNA cleavage. Germline clp1 null zebrafish showed cerebellar neurodegeneration that was rescued by wild-type, but not mutant, human CLP1 expression. Patient-derived induced neurons displayed both depletion of mature tRNAs and accumulation of unspliced pre-tRNAs. Transfection of partially processed tRNA fragments into patient cells exacerbated an oxidative stress-induced reduction in cell survival. Our data link tRNA maturation to neuronal development and neurodegeneration through defective CLP1 function in humans.


Assuntos
Cerebelo/crescimento & desenvolvimento , Cerebelo/patologia , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Proteínas Nucleares/genética , Fosfotransferases/genética , Splicing de RNA , RNA de Transferência/genética , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Fator de Especificidade de Clivagem e Poliadenilação/genética , Feminino , Humanos , Masculino , Camundongos , Modelos Moleculares , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Proteínas Nucleares/metabolismo , Linhagem , Fosfotransferases/metabolismo , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
2.
Mol Cell ; 52(2): 184-92, 2013 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-24095278

RESUMO

In cells, tRNAs are synthesized as precursor molecules bearing extra sequences at their 5' and 3' ends. Some tRNAs also contain introns, which, in archaea and eukaryotes, are cleaved by an evolutionarily conserved endonuclease complex that generates fully functional mature tRNAs. In addition, tRNAs undergo numerous posttranscriptional nucleotide chemical modifications. In Trypanosoma brucei, the single intron-containing tRNA (tRNA(Tyr)GUA) is responsible for decoding all tyrosine codons; therefore, intron removal is essential for viability. Using molecular and biochemical approaches, we show the presence of several noncanonical editing events, within the intron of pre-tRNA(Tyr)GUA, involving guanosine-to-adenosine transitions (G to A) and an adenosine-to-uridine transversion (A to U). The RNA editing described here is required for proper processing of the intron, establishing the functional significance of noncanonical editing with implications for tRNA processing in the deeply divergent kinetoplastid lineage and eukaryotes in general.


Assuntos
Íntrons/genética , Edição de RNA , Splicing de RNA , RNA de Transferência de Tirosina/genética , Trypanosoma brucei brucei/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Endorribonucleases/genética , Endorribonucleases/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Interferência de RNA , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , RNA de Transferência de Tirosina/química , RNA de Transferência de Tirosina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Trypanosoma brucei brucei/metabolismo
3.
Am J Hum Genet ; 99(1): 228-35, 2016 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-27392077

RESUMO

The tRNA splicing endonuclease is a highly evolutionarily conserved protein complex, involved in the cleavage of intron-containing tRNAs. In human it consists of the catalytic subunits TSEN2 and TSEN34, as well as the non-catalytic TSEN54 and TSEN15. Recessive mutations in the corresponding genes of the first three are known to cause pontocerebellar hypoplasia (PCH) types 2A-C, 4, and 5. Here, we report three homozygous TSEN15 variants that cause a milder version of PCH2. The affected individuals showed progressive microcephaly, delayed developmental milestones, intellectual disability, and, in two out of four cases, epilepsy. None, however, displayed the central visual failure seen in PCH case subjects where other subunits of the TSEN are mutated, and only one was affected by the extensive motor defects that are typical in other forms of PCH2. The three amino acid substitutions impacted the protein level of TSEN15 and the stoichiometry of the interacting subunits in different ways, but all resulted in an almost complete loss of in vitro tRNA cleavage activity. Taken together, our results demonstrate that mutations in any known subunit of the TSEN complex can cause PCH and progressive microcephaly, emphasizing the importance of its function during brain development.


Assuntos
Doenças Cerebelares/genética , Endonucleases/genética , Genes Recessivos , Microcefalia/genética , Mutação , Sequência de Aminoácidos , Criança , Pré-Escolar , Endonucleases/química , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Modelos Moleculares , Linhagem
4.
RNA ; 23(4): 567-577, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28096517

RESUMO

Nonsense mutations resulting in a premature stop codon in an open reading frame occur in critical tumor suppressor genes in a large number of the most common forms of cancers and are known to cause or contribute to the progression of disease. Low molecular weight compounds that induce readthrough of nonsense mutations offer a new means of treating patients with genetic disorders or cancers resulting from nonsense mutations. We have identified the nucleoside analog clitocine as a potent and efficacious suppressor of nonsense mutations. We determined that incorporation of clitocine into RNA during transcription is a prerequisite for its readthrough activity; the presence of clitocine in the third position of a premature stop codon directly induces readthrough. We demonstrate that clitocine can induce the production of p53 protein in cells harboring p53 nonsense-mutated alleles. In these cells, clitocine restored production of full-length and functional p53 as evidenced by induced transcriptional activation of downstream p53 target genes, progression of cells into apoptosis, and impeded growth of nonsense-containing human ovarian cancer tumors in xenograft tumor models. Thus, clitocine induces readthrough of nonsense mutations by a previously undescribed mechanism and represents a novel therapeutic modality to treat cancers and genetic diseases caused by nonsense mutations.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Materiais Biomiméticos/farmacologia , Códon sem Sentido/efeitos dos fármacos , Furanos/farmacologia , Nucleosídeos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Nucleosídeos de Pirimidina/farmacologia , Proteína Supressora de Tumor p53/agonistas , Animais , Antimetabólitos Antineoplásicos/síntese química , Antimetabólitos Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/metabolismo , Linhagem Celular Tumoral , Feminino , Furanos/síntese química , Furanos/metabolismo , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Nus , Nucleosídeos/síntese química , Nucleosídeos/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Biossíntese de Proteínas , Nucleosídeos de Pirimidina/síntese química , Nucleosídeos de Pirimidina/metabolismo , Transdução de Sinais , Ativação Transcricional , Carga Tumoral/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Proc Natl Acad Sci U S A ; 113(44): 12508-12513, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27702906

RESUMO

A premature termination codon (PTC) in the ORF of an mRNA generally leads to production of a truncated polypeptide, accelerated degradation of the mRNA, and depression of overall mRNA expression. Accordingly, nonsense mutations cause some of the most severe forms of inherited disorders. The small-molecule drug ataluren promotes therapeutic nonsense suppression and has been thought to mediate the insertion of near-cognate tRNAs at PTCs. However, direct evidence for this activity has been lacking. Here, we expressed multiple nonsense mutation reporters in human cells and yeast and identified the amino acids inserted when a PTC occupies the ribosomal A site in control, ataluren-treated, and aminoglycoside-treated cells. We find that ataluren's likely target is the ribosome and that it produces full-length protein by promoting insertion of near-cognate tRNAs at the site of the nonsense codon without apparent effects on transcription, mRNA processing, mRNA stability, or protein stability. The resulting readthrough proteins retain function and contain amino acid replacements similar to those derived from endogenous readthrough, namely Gln, Lys, or Tyr at UAA or UAG PTCs and Trp, Arg, or Cys at UGA PTCs. These insertion biases arise primarily from mRNA:tRNA mispairing at codon positions 1 and 3 and reflect, in part, the preferred use of certain nonstandard base pairs, e.g., U-G. Ataluren's retention of similar specificity of near-cognate tRNA insertion as occurs endogenously has important implications for its general use in therapeutic nonsense suppression.


Assuntos
Códon sem Sentido/genética , Oxidiazóis/farmacologia , RNA de Transferência/genética , Ribossomos/efeitos dos fármacos , Células HEK293 , Humanos , Biossíntese de Proteínas/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , RNA de Transferência/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Transcrição Gênica/efeitos dos fármacos
7.
Nature ; 447(7140): 87-91, 2007 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-17450125

RESUMO

Nonsense mutations promote premature translational termination and cause anywhere from 5-70% of the individual cases of most inherited diseases. Studies on nonsense-mediated cystic fibrosis have indicated that boosting specific protein synthesis from <1% to as little as 5% of normal levels may greatly reduce the severity or eliminate the principal manifestations of disease. To address the need for a drug capable of suppressing premature termination, we identified PTC124-a new chemical entity that selectively induces ribosomal readthrough of premature but not normal termination codons. PTC124 activity, optimized using nonsense-containing reporters, promoted dystrophin production in primary muscle cells from humans and mdx mice expressing dystrophin nonsense alleles, and rescued striated muscle function in mdx mice within 2-8 weeks of drug exposure. PTC124 was well tolerated in animals at plasma exposures substantially in excess of those required for nonsense suppression. The selectivity of PTC124 for premature termination codons, its well characterized activity profile, oral bioavailability and pharmacological properties indicate that this drug may have broad clinical potential for the treatment of a large group of genetic disorders with limited or no therapeutic options.


Assuntos
Códon sem Sentido/genética , Doenças Genéticas Inatas/tratamento farmacológico , Doenças Genéticas Inatas/genética , Oxidiazóis/farmacologia , Oxidiazóis/uso terapêutico , Biossíntese de Proteínas/efeitos dos fármacos , Alelos , Animais , Disponibilidade Biológica , Distrofina/biossíntese , Distrofina/genética , Doenças Genéticas Inatas/sangue , Humanos , Camundongos , Camundongos Endogâmicos mdx , Oxidiazóis/administração & dosagem , Oxidiazóis/farmacocinética , Fenótipo , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade por Substrato
8.
Drug Discov Today ; 28(1): 103431, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36356786

RESUMO

The concept of using small molecules to therapeutically modulate pre-mRNA splicing was validated with the US Food and Drug Administration (FDA) approval of Evrysdi® (risdiplam) in 2020. Since then, efforts have continued unabated toward the discovery of new splicing-modulating drugs. However, the drug development world has evolved in the 10 years since risdiplam precursors were first identified in high-throughput screening (HTS). Now, new mechanistic insights into RNA-processing pathways and regulatory networks afford increasingly feasible targeted approaches. In this review, organized into classes of biological target, we compile and summarize small molecules discovered, devised, and developed since 2020 to alter pre-mRNA splicing.


Assuntos
Precursores de RNA , Splicing de RNA , Precursores de RNA/genética , Precursores de RNA/metabolismo , Compostos Azo , Pirimidinas , Processamento Alternativo
9.
Nature ; 441(7091): 375-7, 2006 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-16710424

RESUMO

Splicing is required for the removal of introns from a subset of transfer RNAs in all eukaryotic organisms. The first step of splicing, intron recognition and cleavage, is performed by the tRNA-splicing endonuclease, a tetrameric enzyme composed of the protein subunits Sen54, Sen2, Sen34 and Sen15. It has previously been demonstrated that the active sites for cleavage at the 5' and 3' splice sites of precursor tRNA are contained within Sen2 and Sen34, respectively. A recent structure of an archaeal endonuclease complexed with a bulge-helix-bulge RNA has led to the unexpected hypothesis that catalysis requires a critical 'cation-pi sandwich' composed of two arginine residues that serve to position the RNA substrate within the active site. This motif is derived from a cross-subunit interaction between the two catalytic subunits. Here we test the role of this interaction within the eukaryotic endonuclease and show that catalysis at the 5' splice site requires the conserved cation-pi sandwich derived from the Sen34 subunit in addition to the catalytic triad of Sen2. The catalysis of pre-tRNA by the eukaryotic tRNA-splicing endonuclease therefore requires a previously unrecognized composite active site.


Assuntos
Endorribonucleases/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/enzimologia , Archaea/genética , Sítios de Ligação , Cátions/metabolismo , Endorribonucleases/genética , Células Eucarióticas/metabolismo , Modelos Biológicos , Mutação/genética , Precursores de RNA/química , Sítios de Splice de RNA , Splicing de RNA , RNA de Transferência/química , RNA de Transferência de Fenilalanina/genética , RNA de Transferência de Fenilalanina/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
Nat Commun ; 12(1): 3332, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099697

RESUMO

Pre-mRNA splicing is a key controller of human gene expression. Disturbances in splicing due to mutation lead to dysregulated protein expression and contribute to a substantial fraction of human disease. Several classes of splicing modulator compounds (SMCs) have been recently identified and establish that pre-mRNA splicing represents a target for therapy. We describe herein the identification of BPN-15477, a SMC that restores correct splicing of ELP1 exon 20. Using transcriptome sequencing from treated fibroblast cells and a machine learning approach, we identify BPN-15477 responsive sequence signatures. We then leverage this model to discover 155 human disease genes harboring ClinVar mutations predicted to alter pre-mRNA splicing as targets for BPN-15477. Splicing assays confirm successful correction of splicing defects caused by mutations in CFTR, LIPA, MLH1 and MAPT. Subsequent validations in two disease-relevant cellular models demonstrate that BPN-15477 increases functional protein, confirming the clinical potential of our predictions.


Assuntos
Aprendizado Profundo , Marcação de Genes/métodos , Splicing de RNA , Animais , Biologia Computacional , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Éxons , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Proteína 1 Homóloga a MutL/genética , Mutação , Fenetilaminas/administração & dosagem , Piridazinas/administração & dosagem , Esterol Esterase/genética , Transcriptoma , Proteínas tau/genética
11.
Nat Commun ; 12(1): 7299, 2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34911927

RESUMO

Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats in the huntingtin (HTT) gene. Consequently, the mutant protein is ubiquitously expressed and drives pathogenesis of HD through a toxic gain-of-function mechanism. Animal models of HD have demonstrated that reducing huntingtin (HTT) protein levels alleviates motor and neuropathological abnormalities. Investigational drugs aim to reduce HTT levels by repressing HTT transcription, stability or translation. These drugs require invasive procedures to reach the central nervous system (CNS) and do not achieve broad CNS distribution. Here, we describe the identification of orally bioavailable small molecules with broad distribution throughout the CNS, which lower HTT expression consistently throughout the CNS and periphery through selective modulation of pre-messenger RNA splicing. These compounds act by promoting the inclusion of a pseudoexon containing a premature termination codon (stop-codon psiExon), leading to HTT mRNA degradation and reduction of HTT levels.


Assuntos
Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/tratamento farmacológico , Doença de Huntington/genética , Splicing de RNA , Bibliotecas de Moléculas Pequenas/administração & dosagem , Animais , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , Humanos , Doença de Huntington/metabolismo , Camundongos , Splicing de RNA/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , Expansão das Repetições de Trinucleotídeos/efeitos dos fármacos
12.
RNA Biol ; 6(3): 329-34, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19574739

RESUMO

Post-transcriptional regulatory mechanisms, dependent on specific RNA:RNA, RNA:protein, or protein:protein interactions that generate large numbers of different RNP constellations, can have sizeable effects on the expression of any given gene. At the mRNA-specific level, these mechanisms also provide numerous novel targets for small molecule drugs capable of enhancing or inhibiting the accumulation of specific proteins. Here, we describe two drug screening technologies that target the post-transcriptional regulation of specific mRNAs with specific small molecules. In one case the GEMS technology utilizes mRNA-specific 5'- and 3'-UTR pairs to identify compounds that reduce protein production as a consequence of the UTRs. The second example utilizes nonsense-containing mRNAs to identify compounds capable of promoting therapeutic nonsense suppression. Both programs have yielded drug candidates that are presently in clinical testing for human diseases with high unmet clinical needs, thus illustrating the therapeutic potential of targeting post-transcriptional control.


Assuntos
Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica , RNA Mensageiro/genética , Animais , Humanos , Estabilidade de RNA , RNA Mensageiro/metabolismo , Transcrição Gênica
14.
Drug Discov Today ; 12(13-14): 553-60, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17631250

RESUMO

The physiological levels of many clinically important proteins are regulated through cellular mechanisms that control the stability and translational efficiency of mRNA. These post-transcriptional processes, which play a critical role in the regulation of gene expression, depend on interactions of specific trans-acting factors with sequence elements located within the 5'- and 3'-untranslated regions (UTRs) of an mRNA. A technology platform called GEMS (Gene Expression Modulation by Small-molecules) exploits the interactions of UTR elements with the trans-acting factors, thereby specifically targeting mechanisms of post-transcriptional control. In this review we describe how this technology enables the identification of small-molecules that modulate the levels of proteins involved in disease pathogenesis.


Assuntos
Desenho de Fármacos , Regulação da Expressão Gênica , Processamento Pós-Transcricional do RNA , Bibliotecas de Moléculas Pequenas , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Humanos , RNA Mensageiro/genética , RNA Mensageiro/fisiologia , Transativadores/genética , Transativadores/metabolismo
16.
Genes Dev ; 20(8): 939-53, 2006 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-16598037

RESUMO

Transcripts harboring 5' upstream open reading frames (uORFs) are often found in genes controlling cell growth including receptors, oncogenes, or growth factors. uORFs can modulate translation or RNA stability and mediate inefficient translation of these potent proteins under normal conditions. In dysregulated cancer cells, where the gene product, for example Her-2 receptor, is overexpressed, post-transcriptional processes must exist that serve to override the inhibitory effects of the uORFs. The 5' untranslated region (UTR) of Her-2 mRNA contains a short uORF that represses translation of the downstream coding region. We demonstrate that in Her-2 overexpressing breast cancer cells, the 3' UTR of the Her-2 mRNA can override translational inhibition mediated by the Her-2 uORF. Within this 3' UTR, a translational derepression element (TDE) that binds to a 38-kDa protein was identified. These results define a novel biological mechanism in which translational control of genes harboring a 5' uORF can be modulated by elements in their 3' UTRs.


Assuntos
Fases de Leitura Aberta , Processamento Pós-Transcricional do RNA , Receptor ErbB-2/genética , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Humanos , Imunoprecipitação , Microscopia de Fluorescência , Mutagênese , Biossíntese de Proteínas , RNA Mensageiro/genética
17.
Cell ; 117(3): 311-21, 2004 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-15109492

RESUMO

tRNA splicing is a fundamental process required for cell growth and division. The first step in tRNA splicing is the removal of introns catalyzed in yeast by the tRNA splicing endonuclease. The enzyme responsible for intron removal in mammalian cells is unknown. We present the identification and characterization of the human tRNA splicing endonuclease. This enzyme consists of HsSen2, HsSen34, HsSen15, and HsSen54, homologs of the yeast tRNA endonuclease subunits. Additionally, we identified an alternatively spliced isoform of SEN2 that is part of a complex with unique RNA endonuclease activity. Surprisingly, both human endonuclease complexes are associated with pre-mRNA 3' end processing factors. Furthermore, siRNA-mediated depletion of SEN2 exhibited defects in maturation of both pre-tRNA and pre-mRNA. These findings demonstrate a link between pre-tRNA splicing and pre-mRNA 3' end formation, suggesting that the endonuclease subunits function in multiple RNA-processing events.


Assuntos
Endonucleases/química , Endonucleases/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , RNA de Transferência/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Nucléolo Celular/química , Nucléolo Celular/metabolismo , Núcleo Celular/química , Núcleo Celular/metabolismo , Sequência Consenso , Sequência Conservada , Endonucleases/isolamento & purificação , Endorribonucleases/metabolismo , Células HeLa , Humanos , Íntrons , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Subunidades Proteicas , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , RNA Interferente Pequeno/metabolismo , RNA de Transferência/química , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de Aminoácidos
18.
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
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