RESUMEN
To induce RNA interference (RNAi), either small interfering RNAs (siRNAs) are directly introduced into the cell or short hairpin RNAs (shRNAs) are expressed from a DNA vector. At present, shRNAs are commonly synthesized by RNA polymerase III (Pol III) promoters of the H1 and U6 RNAs. In this study, we designed and characterized a new set of plasmid vectors driven by promoters of the Epstein-Barr virus (EBV)-encoded small RNAs (EBERs). The EBERs are the most abundant transcript in infected cells and they are transcribed by Pol III. We showed that the EBER promoters were able to drive the expression of shRNA fusion transcripts. siRNAs processed from these fusion transcripts specifically and effectively inhibited the expression of homologous reporter or endogenous genes in various types of cells. The partial EBER sequences in the fusion transcripts did not activate double-stranded RNA-dependent protein kinase or suppress RNAi. In nasopharyngeal carcinoma cells, the EBER2 promoter was stronger than the H1 and U6 promoters in shRNA synthesis, leading to more effective knockdown of the target genes. Taken together, our findings suggest that the EBER promoters fundamentally different from those of H1 and U6 can be used to drive the intracellular expression of shRNAs for effective silencing of target genes in mammalian cells and particularly, in EBV-infected cells.
Asunto(s)
Terapia Genética/métodos , Herpesvirus Humano 4/genética , Neoplasias Nasofaríngeas/terapia , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño , Línea Celular Tumoral , Estudios de Factibilidad , Expresión Génica , Silenciador del Gen , Ingeniería Genética , Vectores Genéticos/farmacología , Humanos , Neoplasias Nasofaríngeas/metabolismo , ARN Polimerasa III/genética , ARN Mensajero/análisis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
We cloned a major isoform of Xenopus homologue of cold inducible RNA binding protein (CIRP), XCIRP-1. XCIRP-1 was neither cold inducible nor essential for cell division during early embryonic development. Suppression of XCIRP-1 dose dependently produced tailbuds with deformations of the brain and internal organs. The defects were XCIRP-1 specific as they could be rescued by sense transcript. Suppression of XCIRP-1 also disrupted the morphogenetic migration of the C3 blastomeres (lineaged to become the embryonic kidney, the pronephros). In animal cap explants, depletion of XCIRP-1 inhibited activin/retinoic acid induced expressions of pronephros related Xlim-1 and WT1 genes. These results suggest that XCIRP-1 is required for the specification and morphogenetic lineage migration of the pronephros.
Asunto(s)
Riñón/embriología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Xenopus , Xenopus laevis/embriología , Anomalías Múltiples/genética , Animales , División Celular , Clonación Molecular , Embrión no Mamífero/fisiología , Evolución Molecular , Femenino , Riñón/citología , Morfogénesis , Fenotipo , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Supresión Genética , Transcripción Genética , Xenopus laevis/genéticaRESUMEN
The aim of this study was to identify and characterize human and mouse Prx-IV. We identified mouse peroxiredoxin IV (Prx-IV) by virtue of sequence homology to its human ortholog previously called AOE372. Mouse Prx-IV conserves an amino-terminal presequence coding for signal peptide. The amino acid sequences of mature mouse and human Prx-IV share 97.5% identity. Phylogenetic analysis demonstrates that Prx-IV is more closely related to Prx-I/-II/-III than to Prx-V/-VI. Previously, we mapped the mouse Prx-IV gene to chromosome X by analyzing two sets of multiloci genetic crosses. Here we performed further comparative analysis of mouse and human Prx-IV genomic loci. Consistent with the mouse results, human Prx-IV gene localized to chromosome Xp22.135-136, in close proximity to SAT and DXS7178. A bacterial artificial chromosome (BAC) clone containing the complete human Prx-IV locus was identified. The size of 7 exons and the sequences of the splice junctions were confirmed by PCR analysis. We conclude that mouse Prx-IV is abundantly expressed in many tissues. However, we could not detect Prx-IV in the conditioned media of NIH-3T3 and Jurkat cells. Mouse Prx-IV was specifically found in the nucleus-excluded region of cultured mouse cells. Intracellularly, overexpression of mouse Prx-IV prevented the production of reactive oxygen species induced by epidermal growth factor or p53. Taken together, mouse Prx-IV is likely a cytoplasmic or organellar peroxiredoxin involved in intracellular redox signaling.
Asunto(s)
Factor de Crecimiento Epidérmico/antagonistas & inhibidores , Peroxidasas/química , Peroxidasas/fisiología , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Mapeo Cromosómico , Clonación Molecular , Medios de Cultivo Condicionados/metabolismo , Exones , Humanos , Células Jurkat , Ratones , Microscopía Confocal , Datos de Secuencia Molecular , Oxidación-Reducción , Peroxidasas/genética , Peroxirredoxinas , Filogenia , Reacción en Cadena de la Polimerasa , Señales de Clasificación de Proteína , Empalme del ARN , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factores de Tiempo , Distribución Tisular , Cromosoma XRESUMEN
We have identified human and mouse peroxiredoxin V (Prx-V) by virtue of the sequence homologies to yeast peroxisomal antioxidant enzyme PMP20. Prx-V represents the fifth of the six currently known subfamilies of mammalian peroxiredoxins. It is a novel organellar enzyme that has orthologs in bacteria. Biochemically, Prx-V is a thioredoxin peroxidase. One important aspect of p53 function in mammalian cells involves induction of apoptosis likely mediated by redox. We show that overexpression of Prx-V prevented the p53-dependent generation of reactive oxygen species. Likewise, Prx-V inhibited p53-induced apoptosis. Thus, Prx-V is critically involved in intracellular redox signaling.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas de Neoplasias , Peroxidasas/farmacología , Proteína p53 Supresora de Tumor/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN/genética , Expresión Génica , Células HeLa , Humanos , Técnicas In Vitro , Ratones , Datos de Secuencia Molecular , Oxidación-Reducción , Peroxidasas/genética , Peroxidasas/metabolismo , Peroxiredoxina III , Peroxirredoxinas , Filogenia , Especies Reactivas de Oxígeno/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Cell fate determining gene mab-21 regulates the proper establishment of neural cell fate and sensory organ identity in nematode. Mammalian homologs of mab-21 have also been implicated to play critical roles in mid-, hindbrain and craniofacial differentiation. We report here the isolation of a mab-21 homolog, XMab21l2, from Xenopus. We showed that its expression in Xenopus was initiated at gastrulation and prominent signal was detected in neurulating embryos at the neural tube, the optic tissue, the developing midbrain, and the pharyngeal pouches. We demonstrated by RNA interference (RNAi), together with other antisense approaches, that XMab21l2 expression is required for the completion of gastrulation and subsequent neural development.