RESUMEN
The yeast exosome is a complex of 3' --> 5' exoribonucleases. Sequence analysis identified putative human homologues for exosome components, although several were found only as expressed sequence tags. Here we report the cloning of full-length cDNAs, which encode putative human homologues of the Rrp40p, Rrp41p, and Rrp46p components of the exosome. Recombinant proteins were expressed and used to raise rabbit antisera. In Western blotting experiments, these decorated HeLa cell proteins of the predicted sizes. All three human proteins were enriched in the HeLa cells nucleus and nucleolus, but were also clearly detected in the cytoplasm. Size exclusion chromatography revealed that hRrp40p, hRrp41p, and hRrp46p were present in a large complex. This cofractionated with the human homologues of other exosome components, hRrp4p and PM/Scl-100. Anti-PM/Scl-positive patient sera coimmunoprecipitated hRrp40p, hRrp41p, and hRrp46p demonstrating their physical association. The immunoprecipitated complex exhibited 3' --> 5' exoribonuclease activity in vitro. hRrp41p was expressed in yeast and shown to suppress the lethality of genetic depletion of yeast Rrp41p. We conclude that hRrp40p, hRrp41p, and hRrp46p represent novel components of the human exosome complex.
Asunto(s)
Exorribonucleasas/análisis , Animales , Secuencia de Bases , Núcleo Celular/química , Clonación Molecular , Citoplasma/química , Exorribonucleasas/genética , Complejo Multienzimático de Ribonucleasas del Exosoma , Células HeLa , Humanos , Datos de Secuencia Molecular , Peso Molecular , Pruebas de Precipitina , Proteínas de Unión al ARN , ConejosRESUMEN
The structural organization of the hamster gene encoding the intermediate filament (IF) protein desmin has been determined. The gene, 6.5 kb in length, contains nine exons with a total length of 2169 nucleotides. Remarkably, the intervening sequences map at positions that fully correspond to those of the vimentin gene. The derived complete primary structure for hamster desmin (468 amino acids; 53,250 daltons) reveals striking species variations in the NH2-terminal domain of desmin. A plasmid containing the complete transcription unit of the desmin gene was transfected into hamster lens cells and into human epithelial (HeLa) cells. In both nonmuscle cell lines the desmin gene was biologically active. The synthesized desmin assembled into authentic IFs, as monitored by immunofluorescence. Double immunofluorescence staining showed that the newly formed desmin filaments colocalize with preexisting vimentin filaments, but not with preexisting keratin filaments.
Asunto(s)
Citoesqueleto/metabolismo , Desmina/genética , Filamentos Intermedios/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Cricetinae , Desmina/biosíntesis , Genes , Células HeLa , Humanos , Cristalino , Mesocricetus , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Transcripción Genética , Transfección , Vimentina/genéticaAsunto(s)
ADN/análisis , Desmina/genética , Vimentina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Cricetinae , Desmina/análisis , Vimentina/análisisRESUMEN
Recombinant cDNA plasmids for the intermediate filament proteins desmin and vimentin were constructed from baby hamster kidney (BHK-21) mRNA. Analysis of four desmin clones gave a sequence of 1574 nucleotides, which is 75% of the total mRNA length. The derived amino acid sequence for hamster desmin shows 92% overall homology with chicken desmin; the homology with hamster vimentin is highest in the alpha-helical middle part (74%). The 3'-noncoding region of desmin mRNA is found to be 677 nucleotides long. With the aid of 5'- and 3'-specific probes, it has been established that there is a single gene for desmin in the hamster genome. This gene expresses a single mRNA species of 2.2 kilobases. Hybridization experiments of a number of DNAs with desmin and vimentin probes show that there are distinct restriction enzyme fragments carrying vimentin and desmin sequences in the genome of representatives of all vertebrate classes.
Asunto(s)
ADN/metabolismo , Desmina/genética , Vimentina/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Pollos , Cricetinae , Enzimas de Restricción del ADN , Proteína Ácida Fibrilar de la Glía/genética , Riñón , Plásmidos , Conformación Proteica , ARN Mensajero/genética , Especificidad de la EspecieRESUMEN
The structure of the chromosomal gene encoding the intermediate filament protein vimentin is described. This gene, which is present as a single copy in the hamster genome, comprises about 10 kb of DNA and contains more than 80% of intron sequences. S1 mapping and sequence analysis reveal nine exons with a total length of 1848 nucleotides. For the complete primary structure of hamster vimentin, 464 amino acids are predicted, giving a molecular weight of 53,500 daltons. The intron positions are at codons 186, 206/207, 238/239, 292/293, 334/335, 408, 423, and 451/452. The overall homology with chicken desmin is 60% and is even higher in the central (alpha-helical) regions of both molecules. Cross-hybridization at the DNA level, however, is low. Comparison of the amino acid sequence of vimentin with prekeratin sequences shows that there is lesser homology of primary structure, but both the position and size of alpha-helical regions are strongly conserved. At the 5' end of the gene there is a consensus promoter sequence. The first AUG start codon is found 132 nucleotides downstream of the estimated cap site. The 3' nontranslated sequence shows homologies with the chicken vimentin gene. An interesting feature of the vimentin gene is a stretch of 44 nucleotides of alternating dC and dA within intron 2 that may form left-handed Z-DNA.
