RESUMEN
Fine tuning of c-MYC expression is critical for its action and is achieved by several regulatory mechanisms. The contribution of c-myc mRNA regulatory sequences on its translational control has been investigated individually. However, putative interactions have not been addressed so far. The effect of these interactions upon the translatability of monocistronic and bicistronic chimaeric mRNAs, carrying combinations of the c-myc mRNA 5'-untranlated region (UTR), 3'-UTR, and coding region instability element (CRD) was investigated on this study. The presence of the 5'-UTR induced an increase in translatability of 50%. The presence of the CRD element, when in frame, reduced translatability by approximately 50%, regardless of the expression levels of the wild type CRD- binding protein (CRD-BP/IMP1). Conversely, overexpression of a mutated CRD-BP/IMP1 (Y396F) further impeded translation of the chimaeric mRNAs carrying its cognate sequences. The presence of the c-myc 3'-UTR increased translatability by approximately 300% affecting both cap and c-myc internal ribosome entry site (IRES) mediated translation. In addition, 3'-UTR rescued the cap mediated translation in the presence of the polyadenylation inhibitor cordycepin. Furthermore, the 3'-UTR rescued cap mediated translation under metabolic stress conditions and this was enhanced in the absence of a long poly (A) tail.
Asunto(s)
Sitios Internos de Entrada al Ribosoma/fisiología , Biosíntesis de Proteínas/fisiología , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Western Blotting , Genes myc/genética , Células HEK293 , Humanos , Mutagénesis , Proteínas Proto-Oncogénicas c-myc/genética , Estrés FisiológicoRESUMEN
Poly(A)polymerase-alpha (PAPOLA) has been the most extensively investigated mammalian polyadenylating enzyme, mainly in regard to its multifaceted post-translational regulation. The possibility of translational regulation of this enzyme was addressed. The transcription start site was mapped and two uORFs, highly conserved among several species, were identified in the 211-bp long, GC-rich, 5' UTR of the PAPOLA mRNA. Mutation of the 5' proximal AUG resulted in increased translational efficiency of the adjacent coding sequence, whereas no significant effect was observed after mutation of the second AUG. These observations imply that translational regulation is among the conserved mechanisms regulating PAPOLA expression.
Asunto(s)
Regiones no Traducidas 5' , Regulación Enzimológica de la Expresión Génica , Polinucleotido Adenililtransferasa/genética , ARN Mensajero/metabolismo , Transcripción Genética , Secuencia de Bases , Línea Celular , Secuencia Conservada , Exones , Secuencia Rica en GC , Humanos , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta , ARN Mensajero/química , Sitio de Iniciación de la Transcripción , TransfecciónRESUMEN
The coding region determinant-binding protein (CRD-BP) is an RNA binding protein that recognizes c-myc and IGF-II leader 3 mRNAs as well as the oncofetal H19 RNA. CRD-BP exhibits an oncofetal pattern of expression and has been detected in the majority of colon (81%), breast (58.5%) and sarcoma (73%) tumors. The study of CRD-BP expression was extended in brain tumors and Non small cell lung (NSCL) carcinomas and 12/24 malignant, 2/5 benign neuroepithelial tumors and 4/15 of NSCL carcinomas were found positive. All normal matching tissues tested were found negative. The highest frequency (60%) of CRD-BP positive tumors was observed in meningiomas, either benign (11/18) or atypical (3/3). These findings confirm that CRD-BP expression is restricted in tumors; the frequency of its de novo expression may vary according to tumor type and appears to be an early event in the transformation process.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Unión al ARN/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Biomarcadores de Tumor/genética , Encéfalo/metabolismo , Neoplasias Encefálicas/genética , Carcinoma de Células Grandes/genética , Carcinoma de Células Grandes/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Estudios de Casos y Controles , Humanos , Pulmón/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/metabolismo , Meningioma/genética , Meningioma/metabolismo , Proteínas de Unión al ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The oncofetal CRD-BP/IMP1 RNA binding protein regulates posttranscriptionally a handful of RNA transcripts, implicated in cell adhesion and invadopodia formation and was recently identified as a target of the beta-catenin/Tcf transcription factor that is constitutively activated in colorectal carcinomas (CRCs). The expression of CRD-BP/IMP1 was studied in normal adult intestines and CRCs. In normal mucosa, CRD-BP/IMP1 immunoreactivity was observed in few scattered cells located predominantly at or near the bottom of the crypts, whereas in CRCs the protein was detectable in tumor cells of 50% of the specimens analyzed. CRD-BP/IMP1 mRNA expression was measured by qRT-PCR in 78 CRCs. Thirty-two (41%) of the specimens were negative or had negligible expression, whereas the remaining forty-six (59%) expressed a wide range of CRD-BP/IMP1 mRNA levels. CRD-BP/IMP1 mRNA expression correlated with that of the putative stem/progenitor cell marker Musashi-1 mRNA (p = 0. 035). CRD-BP/IMP1 positive tumors metastasized and/or recurred more frequently (p = 0.001) and its expression defined a group of patients with shorter survival (p = 0.014). Furthermore, in a multivariate analysis CRD-BP/IMP1 expression was found to be an independent predictor of survival (p = 0.015). For stage I & II patients, the differences in metastasis/recurrence and survival rates remained significant (p = 0.001 and 0.033, respectively). These findings indicate that CRD-BP/IMP1 positive tumors exhibit early disease dissemination and unfavorable prognosis.