E2F1 activates p53 transcription through its distal site and participates in apoptosis induction in HPV-positive cells

AbstractThe p53 tumor suppressor protein, one of the most extensively studied proteins, plays a pivotal role in cellular checkpoints that respond to DNA damage to prevent tumorigenesis. However, the transcriptional control of the p53 gene has not been fully characterized. We report that the transcription factor E2F1 binds only to the E2F1 distal site of the p53 promoter in the human papillomavirus positive carcinoma HeLa cell line. Moreover, we showed that etoposide, a DNA damaging agent, activates p53 transcription through the E2F1 pathway. This increase correlates with apoptosis induction as disruption of this pathway led to reduced apoptosis stimulation by the DNA damaging agent

Key contribution of eIF4H-mediated translational control in tumor promotion.

Dysregulated expression of translation initiation factors has been associated with carcinogenesis, but underlying mechanisms remains to be fully understood. Here we show that eIF4H (eukaryotic translation initiation factor 4H), an activator of the RNA helicase eIF4A, is overexpressed in lung carcinomas and predictive of response to chemotherapy. In lung cancer cells, depletion of eIF4H enhances sensitization to chemotherapy, decreases cell migration and inhibits tumor growth in vivo, in association with reduced translation of mRNA encoding cell-proliferation (c-Myc, cyclin D1) angiogenic (FGF-2) and anti-apoptotic factors (CIAP-1, BCL-xL). Conversely, each isoform of eIF4H acts as an oncogene in NIH3T3 cells by stimulating transformation, invasion, tumor growth and resistance to drug-induced apoptosis together with increased translation of IRES-containing or structured 5'UTR mRNAs. These results demonstrate that eIF4H plays a crucial role in translational control and can promote cellular transformation by preferentially regulating the translation of potent growth and survival factor mRNAs, indicating that eIF4H is a promising new molecular target for cancer therapy

An upstream open reading frame within an IRES controls expression of a specific VEGF-A isoform

Vascular endothelial growth factor A (VEGF-A) is a potent secreted mitogen critical for physiological and pathological angiogenesis. Regulation of VEGF-A occurs at multiple levels, including transcription, mRNA stabilization, splicing, translation and differential cellular localization of various isoforms. Recent advances in our understanding of the posttranscriptional regulation of VEGF-A are comprised of the identification of stabilizing mRNA-binding proteins and the discovery of two internal ribosomal entry sites (IRES) as well as two alternative initiation codons in the 5′UTR of the VEGF-A mRNA. We have previously reported that VEGF-A translation initiation at both the AUG and CUG codons is dependent on the exon content of the coding region. In this report, we show that the expression of different VEGF-A isoforms is regulated by a small upstream open reading frame (uORF) located within an internal ribosome entry site, which is translated through a cap-independent mechanism. This uORF acts as a cis-regulatory element that regulates negatively the expression of the VEGF 121 isoform. Our data provide a framework for understanding how VEGF-A mRNAs are translated, and how the production of the VEGF 121 isoform is secured under non-hypoxic environmental conditions

Alternative-splicing-based bicistronic vectors for ratio-controlled protein expression and application to recombinant antibody production

In the last decade polycistronic vectors have become essential tools for both basic science and gene therapy applications. In order to co-express heterologous polypeptides, different systems have been developed from Internal Ribosome Entry Site (IRES) based vectors to the use of the 2A peptide. Unfortunately, these methods are not fully suitable for the efficient and reproducible modulation of the ratio between the proteins of interest. Here we describe a novel bicistronic vector type based on the use of alternative splicing. By modifying the consensus sequence that governs splicing, we demonstrate that the ratio between the synthesized proteins could easily vary from 1 : 10 to 10 : 1. We have established this system with luciferase genes and we extended its application to the production of recombinant monoclonal antibodies. We have shown that these vectors could be used in several typical cell lines with similar efficiencies. We also present an adaptation of these vectors to hybrid alternative splicing/IRES constructs that allow a ratio-controlled expression of proteins of interest in stably transfected cell lines

Rôle d'elF4H dans la traduction et le développement tumoral

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Etude du contrôle de la traduction des isoformes du VEFG-A

Le VEGF A est un facteur angiogénique majeur intervenant dans l'angiogenèse physiologique et pathologique. Son expression est régulée à toutes les étapes de l'expression génique.. Le pré-ARNm est soumis à un épissage alternatif qui permet la synthèse de trois isoformes majeures de 121, 165 et 189 acides aminés. L'ARNm du VEGF A possède deux sites d'entrée interne des ribosomes (IRES) et deux codons alternatifs d'initiation de la traduction. L'initiation de la traduction au codon AUG, sous le contrôle de l'IRES A, entraîne la synthèse de VEGF A sécrété. Le codon CUG, sous le contrôle de l'IRES B, est responsable de la synthèse du L-VEGF. Cette isoforme est maturée par clivage pour générer une molécule de VEGF A sécrétée et un fragment amino-terminal intracellulaire (le N-VEGF). Nous avons voulu identifier par quel mécanisme la séquence exonique du VEGF, issue de l'épissage alternatif, contrôle l'initiation de la traduction. Nous avons mis en évidence une séquence en amont de l'AUG impliquée dans cette régulation. Cette séquence contenant une uORF, nous avons examiné l'effet de sa traduction sur l'expression du VEGF et le mécanisme de traduction de cette uORF. Nos résultats montrent que cette uORF est fonctionnelle et qu'elle est impliquée dans la régulation de l'expression des isoformes du VEGF. Cette uORF est traduite par un mécanisme indépendant de la coiffe et inhibe la synthèse de l'isoforme 121 dans des conditions environnementales non-hypoxiques. L'ensemble de cette étude permet de mieux comprendre les mécanismes de synthèse des différentes isoformes du VEGF dans des processus pathologiques.TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Etude in vitro et in vivo de l'expression du VEGF-A (Vascular Endothelial Growth Factor-A) / Stéphanie Bornes ; Hervé Prats, Christian Touriol

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Alternative Translation Initiation of Human Fibroblast Growth Factor 2 mRNA Controlled by Its 3′-Untranslated Region Involves a Poly(A) Switch and a Translational Enhancer

International audienceFive fibroblast growth factor 2 (FGF-2) isoforms are synthesized from human FGF-2 mRNA by a process of alternative initiation of translation. The regulation of FGF-2 isoform expression by the mRNA 5823-nucleotide-long 3′-untranslated region containing eight alternative polyadenylation sites was examined. Because previous studies had shown that FGF-2 expression was regulated in primary cells but not in transformed cells, primary human skin fibroblasts were used in this study. Using an approach of cell transfection with synthetic reporter mRNAs, a novel translational enhancer (3′-TE) was identified in the 1370-nucleotide mRNA segment located upstream from the eighth poly(A) site. Deletion mutagenesis showed that the 3′-TE was composed of two domains with additive effects. The 3′-TE exhibited the unique feature of modulating the use of FGF-2 alternative initiation codons, which favored the relative expression of CUG-initiated isoforms. Interestingly, the use of an alternative polydenylation site removing the 3′-TE was detected in skin fibroblasts in response to heat shock and cell density variations. At high cell densities, 3′-TE removal was correlated with a loss of CUG-initiated FGF-2 expression. These data show that the FGF-2 mRNA 3′-untranslated region is able to modulate FGF-2 isoform expression by the coupled processes of translation activation and alternative polyadenylation

The StkP/PhpP Signaling Couple in Streptococcus pneumoniae: Cellular Organization and Physiological Characterization ▿ †

In Streptococcus pneumoniae, stkP and phpP, encoding the eukaryotic-type serine-threonine kinase and PP2C phosphatase, respectively, form an operon. PhpP has the features of a so-called “soluble” protein, whereas StkP protein is membrane associated. Here we provide the first genetic and physiological evidence that PhpP and StkP, with antagonist enzymatic activities, constitute a signaling couple. The StkP-PhpP couple signals competence upstream of the competence-specific histidine kinase ComD, receptor for the oligopeptide pheromone “competence stimulating peptide.” We show that PhpP activity is essential in a stkP+ genetic background, suggesting tight control of StkP activity by PhpP. Proteins PhpP and StkP colocalized to the cell membrane subcellular fraction and likely belong to the same complex, as revealed by coimmunoprecipitation in cellular extracts. Specific coimmunoprecipitation of the N-kinase domain of StkP and PhpP recombinant proteins by PhpP-specific antibodies demonstrates direct interaction between these proteins. Consistently, flow cytometry analysis allowed the determination of the cytoplasmic localization of PhpP and of the N-terminal kinase domain of StkP, in contrast to the periplasmic localization of the StkP C-terminal PASTA (penicillin-binding protein and serine-threonine kinase associated) domain. A signaling route involving interplay between serine, threonine, and histidine phosphorylation is thus described for the first time in this human pathogen