RESUMO
Nucleocytoplasmic transport of mRNA is essential for eukaryotic gene expression. However, how mRNA is exported from the nucleus is mostly unknown. To elucidate the mechanisms of mRNA transport, we took a genetic approach to identify genes, the products of which play a role in that process. From about 1000 temperature -sensitive (ts- or cs-) mutants, we identified five ts- mutants that are defective in poly(A)+ RNA transport by using a situ hybridization with an oligo(dT)50 as a probe. These mutants accumulate poly(A)+ RNA in the nuclei when shifted to a nonpermissive temperature. All five mutations are tightly linked to the ts- growth defects, are recessive, and fall into four different groups designated as ptr 1-4 (poly(A)+ RNA transport). Interestingly, each group of mutants has a differential localization pattern of poly(A)+ RNA in the nuclei at the nonpermissive temperature, suggesting that they have defects at different steps of the mRNA transport pathway. Localization of a nucleoplasmin-green fluorescent protein fusion suggests that ptr2 and ptr3 have defects also in nuclear protein import. Among the isolated mutants, only ptr2 showed a defect in pre-mRNA splicing. We cloned the ptr2+ and ptr3+ genes and found that they encode Schizosaccharomyces pombe homologues of the mammalian RCC1, a guanine nucleotide exchange factor for RAN/TC4, and the ubiquitin-activating enzyme E1 involved in ubiquitin conjugation, respectively. The ptr3+ gene is essential for cell viability, and Ptr3p tagged with green fluorescent protein was localized in both the nucleus and the cytoplasm. This is the first report suggesting that the ubiquitin system plays a role in mRNA export.
Assuntos
Proteínas de Transporte/genética , Proteínas de Membrana/genética , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Schizosaccharomyces/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Clonagem Molecular , DNA Fúngico , Genes Fúngicos , Ligases/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutagênese , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleoplasminas , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Poli A/metabolismo , Precursores de RNA , Splicing de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe , Homologia de Sequência de Aminoácidos , Enzimas Ativadoras de Ubiquitina , Ubiquitina-Proteína LigasesRESUMO
Transport of mRNA from the nucleus to the cytoplasm is one of the important steps in gene expression in eukaryotic cells. To elucidate a mechanism of mRNA export, we identified a novel ptr [poly(A)+ RNA transport] mutation, ptr6, which causes accumulation of mRNA in the nucleus and inhibition of growth at the nonpermissive temperature. The ptr6(+) gene was found to encode an essential protein of 393 amino acids, which shares significant homology in amino acid sequence with yTAFII67 of budding yeast Saccharomyces cerevisiae and human hTAFII55, a subunit of the general transcription factor complex TFIID. A Ptr6p-GFP fusion protein is localized in the nucleus, suggesting that Ptr6p functions there. Northern blot analysis using probes for 10 distinct mRNAs showed that the amount of tbp+ mRNA encoding the TATA-binding protein is increased five- to sixfold, whereas amounts of others are rapidly decreased at the nonpermissive temperature in ptr6-1. ptr6 has no defects in nuclear import of an NLS-GFP fusion protein. These results suggest that Ptr6p required for mRNA transport is a Schizosaccharomyces pombe homologue of yTAFII67 and hTAFII55. This is the first report suggesting that a TAF is involved in the nucleocytoplasmic transport of mRNA in addition to the transcription of the protein-coding genes.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Fatores Associados à Proteína de Ligação a TATA , Fator de Transcrição TFIID , Fatores de Transcrição , Citometria de Fluxo , Expressão Gênica , Genes Reguladores , Humanos , Dados de Sequência Molecular , RNA Mensageiro/análise , Saccharomyces cerevisiae/genética , Temperatura , Fatores de Tempo , Transativadores/metabolismo , Transcrição GênicaRESUMO
Cytokine-induced neutrophil chemoattractant (CINC)-1, a counterpart of the human growth-regulated gene product (GRO) of the interleukin-8 family, has been suggested to play critical roles as a mediator of inflammatory reactions with neutrophil infiltration in rats. NF-kappaB has been speculated to be involved in the production of CINC-1, since the NF-kappaB-binding domain is important for the CINC-1 promoter activity in several of our reporter assays. In the present study, we examined the effects of an overexpression of IkappaBalpha, a specific natural inhibitor of NF-kappaB, on CINC-1 production. For this purpose, we constructed two recombinant adenoviruses, AxCAIkappaBalpha and AxCAmutantIkappaBalpha, which express respectively wild IkBa and a mutated nondegradable IkappaBalpha in which serine residues 32 and 36 are replaced by alanine residues. Transfecting wild and mutant IkBa by these adenovirus vectors inhibited NF-kappaB activation and CINC-1 production, which were both caused by IL-1beta stimulation in the normal rat kidney epithelial cell line NRK-52E. We also showed that the nondegradable mutant IkappaBalpha was approximately 30 times more potent than the wild type in inhibiting CINC-1 production. These findings demonstrate that CINC-1 production with NF-kappaB activation is primarily regulated by non-phosphorylated IkBa in the cytoplasm.