RESUMEN
The Epstein-Barr nuclear antigen 3C (EBNA3C), one of the essential latent antigens for Epstein-Barr virus (EBV)-induced immortalization of primary human B lymphocytes in vitro, has been implicated in regulating cell proliferation and anti-apoptosis via interaction with several cellular and viral factors. Gemin3 (also named DDX20 or DP103) is a member of DEAD RNA helicase family which exhibits diverse cellular functions including DNA transcription, recombination and repair, and RNA metabolism. Gemin3 was initially identified as a binding partner to EBNA2 and EBNA3C. However, the mechanism by which EBNA3C regulates Gemin3 function remains unclear. Here, we report that EBNA3C directly interacts with Gemin3 through its C-terminal domains. This interaction results in increased stability of Gemin3 and its accumulation in both B lymphoma cells and EBV transformed lymphoblastoid cell lines (LCLs). Moreover, EBNA3C promotes formation of a complex with p53 and Gemin3 which blocks the DNA-binding affinity of p53. Small hairpin RNA based knockdown of Gemin3 in B lymphoma or LCL cells remarkably attenuates the ability of EBNA3C to inhibit the transcription activity of p53 on its downstream genes p21 and Bax, as well as apoptosis. These findings provide the first evidence that Gemin3 may be a common target of oncogenic viruses for driving cell proliferation and anti-apoptotic activities.
Asunto(s)
Antígenos Virales/metabolismo , Apoptosis/fisiología , Proteína 20 DEAD-Box/metabolismo , Infecciones por Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Antígenos Virales/genética , Linfocitos B/metabolismo , Linfocitos B/virología , Western Blotting , Línea Celular Tumoral , Proteína 20 DEAD-Box/genética , Infecciones por Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Herpesvirus Humano 4/genética , Humanos , Inmunoprecipitación , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The SMN complex mediates the assembly of heptameric Sm protein rings on small nuclear RNAs (snRNAs), which are essential for snRNP function. Specific Sm core assembly depends on Sm proteins and snRNA recognition by SMN/Gemin2- and Gemin5-containing subunits, respectively. The mechanism by which the Sm proteins are gathered while preventing illicit Sm assembly on non-snRNAs is unknown. Here, we describe the 2.5 Å crystal structure of Gemin2 bound to SmD1/D2/F/E/G pentamer and SMN's Gemin2-binding domain, a key assembly intermediate. Remarkably, through its extended conformation, Gemin2 wraps around the crescent-shaped pentamer, interacting with all five Sm proteins, and gripping its bottom and top sides and outer perimeter. Gemin2 reaches into the RNA-binding pocket, preventing RNA binding. Interestingly, SMN-Gemin2 interaction is abrogated by a spinal muscular atrophy (SMA)-causing mutation in an SMN helix that mediates Gemin2 binding. These findings provide insight into SMN complex assembly and specificity, linking snRNP biogenesis and SMA pathogenesis.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas del Complejo SMN/metabolismo , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas de Unión al ARN/genética , Alineación de SecuenciaRESUMEN
RNAs in cells are associated with RNA-binding proteins (RBPs) to form ribonucleoprotein (RNP) complexes. The RBPs influence the structure and interactions of the RNAs and play critical roles in their biogenesis, stability, function, transport and cellular localization. Eukaryotic cells encode a large number of RBPs (thousands in vertebrates), each of which has unique RNA-binding activity and protein-protein interaction characteristics. The remarkable diversity of RBPs, which appears to have increased during evolution in parallel to the increase in the number of introns, allows eukaryotic cells to utilize them in an enormous array of combinations giving rise to a unique RNP for each RNA. In this short review, we focus on the RBPs that interact with pre-mRNAs and mRNAs and discuss their roles in the regulation of post-transcriptional gene expression.
Asunto(s)
Regulación de la Expresión Génica , Ribonucleoproteínas/metabolismo , Animales , HumanosRESUMEN
The cytochrome P450 (Cyp) 2a5 gene can be upregulated transcriptionally or by mRNA stabilization. The heterogeneous nuclear ribonucleoprotein (hnRNP) A1 interacting with the CYP2A5 mRNA has been shown to be a key post-transcriptional regulator of the Cyp2a5 gene. The aim of this study was to investigate if the transcriptional and post-transcriptional steps of Cyp2a5 expression are linked. This was done by modifying the transcription rate with transcriptional inducers (phenobarbital and cyclic AMP) and inhibitors (actinomycin D and 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole) and analyzing the effects upon post-transcriptional events. We found that inhibition of transcription led to relocalization of hnRNP A1 from the nucleus to the cytoplasm, to its strongly increased binding to the cytoplasmic CYP2A5 mRNA and to CYP2A5 mRNA stabilization. In contrast, stimulated transcription resulted in increased binding of nuclear hnRNP A1 to the Cyp2a5 promoter, and overexpression of hnRNP A1 led to stimulated transcription of a Cyp2a5 promoter-driven luciferase recombinant. This strongly suggests that the transcriptional and post-transcriptional stages of Cyp2a5 expression are interrelated and that the nucleocytoplasmic shuttling hnRNP A1 may coordinate these different steps.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/genética , Regulación Enzimológica de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Oxigenasas de Función Mixta/genética , Regiones Promotoras Genéticas/fisiología , Transcripción Genética , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Secuencia de Bases , Citocromo P-450 CYP2A6 , Familia 2 del Citocromo P450 , ADN/análisis , Genes Reporteros , Masculino , Ratones , Ratones Endogámicos DBA , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Estabilidad del ARN , ARN Mensajero/metabolismoRESUMEN
Our previous evidence suggests that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 plays a part in the regulation of the Cyp2a5 gene by interacting with the 3' untranslated region (UTR) of the CYP2A5 mRNA. However, the exact role of this interaction is not clear. The aim of the present work was to gain further insight into the regulation process of Cyp2a5. For this purpose the 3' UTR of CYP2A5 was fused to the coding region of luciferase mRNA. Luciferase recombinants containing either the full length 3' UTR, or the 3' UTR lacking a previously described 71 nucleotide (nt) region (the hnRNP A1 primary binding site), were transiently expressed in cells expressing or lacking hnRNP A1. The expression of the luciferase recombinants was examined both at mRNA and enzyme activity levels. The results disclosed that the presence of hnRNP A1 was required for the high expression of the recombinant carrying the full length 3' UTR of CYP2A5. Deletion of the hnRNP A1 primary binding site dramatically modified the expression pattern: the mRNA levels and luciferase activities of the deletion mutant were independent from hnRNP A1. These results conclusively demonstrate that the 71 nt region in the 3' UTR of CYP2A5 mRNA can confer hnRNP A1-dependent regulation to a gene. In addition, comparison of RNA levels and luciferase activities suggested that regions flanking the hnRNP A1 binding site could regulate translation of the CYP2A5 mRNA. These results are consistent with a model in which the binding of hnRNP A1 to the 71 nt putative hairpin-loop region in the CYP2A5 mRNA 3' UTR upregulates mRNA levels possibly by protecting the mRNA from degradation.
Asunto(s)
Regiones no Traducidas 3'/metabolismo , Hidrocarburo de Aril Hidroxilasas/genética , Regulación de la Expresión Génica/fisiología , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Oxigenasas de Función Mixta/genética , ARN Mensajero/metabolismo , Regiones no Traducidas 3'/genética , Animales , Sitios de Unión/genética , Sitios de Unión/fisiología , Northern Blotting , Western Blotting , Línea Celular , Citocromo P-450 CYP2A6 , Familia 2 del Citocromo P450 , Genes Reporteros , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Luciferasas/genética , Ratones , ARN/genética , ARN/metabolismo , ARN Mensajero/genética , Eliminación de Secuencia , TransfecciónRESUMEN
The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) functions in the packaging of nascent RNA polymerase II transcripts and participates in a variety of nuclear and cytoplasmic processes that modulate gene expression. The RNA binding characteristics of hnRNP A1 suggest that it can modulate the expression of specific genes, but little is known about its possible targets in vivo. In this article, we show that hnRNP A1 interacts with the transcript of a cytochrome P450 gene, Cyp2a5, induced by xenobiotics and during liver damage. Binding of the hnRNP A1 to CYP2A5 mRNA was demonstrated by immunoprecipitation of the xenobiotic-stimulated (37/39 kDa) CYP2A5 mRNA-protein complex with a monoclonal anti-hnRNP A1 antibody, by partial trypsin digestion of the complex, and by showing that the RNA-protein complex is not formed with protein extracts from cells lacking the hnRNP A1. We also show that a specific hepatotoxic inducer of the Cyp2a5 gene, pyrazole, increases the cytoplasmic levels of hnRNP A1 in vivo. Finally, we show that hnRNP A1 can be overexpressed in mouse primary hepatocytes, leading to an accumulation of the CYP2A5 mRNA. Collectively, these results indicate that the hnRNP A1 is an important regulator of the Cyp2a5 gene.