scholarly journals c-myc Internal Ribosome Entry Site Activity Is Developmentally Controlled and Subjected to a Strong Translational Repression in Adult Transgenic Mice

2001 ◽  
Vol 21 (5) ◽  
pp. 1833-1840 ◽  
Author(s):  
Laurent Créancier ◽  
Pascale Mercier ◽  
Anne-Catherine Prats ◽  
Dominique Morello
Keyword(s):  
Cell Proliferation ◽  
Transgenic Mice ◽  
Internal Ribosome Entry Site ◽  
Ex Vivo ◽  
Cell Types ◽  
Tumor Formation ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Internal Ribosome Entry

ABSTRACT The expression of c-myc proto-oncogene, a key regulator of cell proliferation and apoptosis, is controlled at different transcriptional and posttranscriptional levels. In particular, the c-myc mRNA contains an internal ribosome entry site (IRES) able to promote translation initiation independently from the classical cap-dependent mechanism. We analyzed the variations of c-myc IRES activity ex vivo in different proliferating cell types, and in vivo in transgenic mice expressing a bicistronic dual luciferase construct. c-myc IRES efficiency was compared to that of encephalomyocarditis virus (EMCV) IRES under the same conditions. The c-myc IRES was active but with variable efficiency in all transiently transfected cell types; it was also active in the 11-day- old (E11) embryo and in some tissues of the E16 embryo. Strikingly, its activity was undetected or very low in all adult organs tested. In contrast, EMCV IRES was very active in most cell types ex vivo, as well as in embryonic and adult tissues. These data suggest a crucial role of IRES in the control of c-mycgene expression throughout development, either during embryogenesis where its activity might participate in cell proliferation or later on, where its silencing could contribute to the downregulation of c-myc expression, whose deregulation leads to tumor formation.

scholarly journals Fibroblast Growth Factor 2 Internal Ribosome Entry Site (Ires) Activity Ex Vivo and in Transgenic Mice Reveals a Stringent Tissue-Specific Regulation

2000 ◽  
Vol 150 (1) ◽  
pp. 275-281 ◽  
Author(s):  
Laurent Créancier ◽  
Dominique Morello ◽  
Pascale Mercier ◽  
Anne-Catherine Prats
Keyword(s):  
Growth Factor ◽  
Transgenic Mice ◽  
Internal Ribosome Entry Site ◽  
Tissue Specificity ◽  
Ex Vivo ◽  
Fibroblast Growth Factor 2 ◽  
Fibroblast Growth ◽  
Entry Site ◽  
Internal Ribosome Entry

Fibroblast growth factor 2 (FGF-2) is a powerful mitogen involved in proliferation, differentiation, and survival of various cells including neurons. FGF-2 expression is translationally regulated; in particular, the FGF-2 mRNA contains an internal ribosome entry site (IRES) allowing cap-independent translation. Here, we have analyzed FGF-2 IRES tissue specificity ex vivo and in vivo by using a dual luciferase bicistronic vector. This IRES was active in most transiently transfected human and nonhuman cell types, with a higher activity in p53 −/− osteosarcoma and neuroblastoma cell lines. Transgenic mice were generated using bicistronic transgenes with FGF-2 IRES or encephalomyocarditis virus (EMCV) IRES. Measurements of luciferase activity revealed high FGF-2 IRES activity in 11-d-old embryos (E11) but not in the placenta; activity was high in the heart and brain of E16. FGF-2 IRES activity was low in most organs of the adult, but exceptionally high in the brain. Such spatiotemporal variations were not observed with the EMCV IRES. These data, demonstrating the strong tissue specificity of a mammalian IRES in vivo, suggest a pivotal role of translational IRES- dependent activation of FGF-2 expression during embryogenesis and in adult brain. FGF-2 IRES could constitute, thus, a powerful tool for gene transfer in the central nervous system.

scholarly journals Novel Fluorescence-Based Screen To Identify Small Synthetic Internal Ribosome Entry Site Elements

2001 ◽  
Vol 21 (8) ◽  
pp. 2826-2837 ◽  
Author(s):  
Arun Venkatesan ◽  
Asim Dasgupta
Keyword(s):  
Internal Ribosome Entry Site ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Independent Manner ◽  
Internal Ribosome Entry

ABSTRACT We report here a novel fluorescent protein-based screen to identify small, synthetic internal ribosome entry site (IRES) elements in vivo. A library of bicistronic plasmids encoding the enhanced blue and green fluorescent proteins (EBFP and EGFP) separated by randomized 50-nucleotide-long sequences was amplified in bacteria and delivered into mammalian cells via protoplast fusion. Cells that received functional IRES elements were isolated using the EBFP and EGFP reporters and fluorescence-activated cell sorting, and several small IRES elements were identified. Two of these elements were subsequently shown to possess IRES activity comparable to that of a variant of the encephalomyocarditis virus IRES element in a context-independent manner both in vitro and in vivo, and these elements functioned in multiple cell types. Although no sequence or structural homology was apparent between the synthetic IRES elements and known viral and cellular IRES elements, the two synthetic IRES elements specifically blocked poliovirus (PV) IRES-mediated translation in vitro. Competitive protein-binding experiments suggested that these IRES elements compete with PV IRES-mediated translation by utilizing some of the same factors as the PV IRES to direct translation. The utility of this fluorescent protein-based screen in identifying IRES elements with improved activity as well as in probing the mechanism of IRES-mediated translation is discussed.

scholarly journals Absence of Internal Ribosome Entry Site-Mediated Tissue Specificity in the Translation of a Bicistronic Transgene

1999 ◽  
Vol 73 (4) ◽  
pp. 2729-2738 ◽  
Author(s):  
Chloë Shaw-Jackson ◽  
Thomas Michiels
Keyword(s):  
Internal Ribosome Entry Site ◽  
Complex Structure ◽  
Cell Types ◽  
Transcription Unit ◽  
Theiler's Virus ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Initiation Of Translation

ABSTRACT The 5′ noncoding regions of the genomes of picornaviruses form a complex structure that directs cap-independent initiation of translation. This structure has been termed the internal ribosome entry site (IRES). The efficiency of translation initiation was shown, in vitro, to be influenced by the binding of cellular factors to the IRES. Hence, we hypothesized that the IRES might control picornavirus tropism. In order to test this possibility, we made a bicistronic construct in which translation of the luciferase gene is controlled by the IRES of Theiler’s murine encephalomyelitis virus. In vitro, we observed that the IRES functions in various cell types and in macrophages, irrespective of their activation state. In vivo, we observed that the IRES is functional in different tissues of transgenic mice. Thus, it seems that the IRES is not an essential determinant of Theiler’s virus tropism. On the other hand, the age of the mouse could be critical for IRES function. Indeed, the IRES was found to be more efficient in young mice. Picornavirus IRESs are becoming popular tools in transgenesis technology, since they allow the expression of two genes from the same transcription unit. Our results show that the Theiler’s virus IRES is functional in cells of different origins and that it is thus a broad-spectrum tool. The possible age dependency of the IRES function, however, could be a drawback for gene expression in adult mice.

scholarly journals La Autoantigen Is Necessary for Optimal Function of the Poliovirus and Hepatitis C Virus Internal Ribosome Entry Site In Vivo and In Vitro

2004 ◽  
Vol 24 (15) ◽  
pp. 6861-6870 ◽  
Author(s):  
Mauro Costa-Mattioli ◽  
Yuri Svitkin ◽  
Nahum Sonenberg
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosome Entry Site ◽  
Ribosomal Subunit ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Entry Site ◽  
Internal Ribosome Entry

ABSTRACT Translation of poliovirus and hepatitis C virus (HCV) RNAs is initiated by recruitment of 40S ribosomes to an internal ribosome entry site (IRES) in the mRNA 5′ untranslated region. Translation initiation of these RNAs is stimulated by noncanonical initiation factors called IRES trans-activating factors (ITAFs). The La autoantigen is such an ITAF, but functional evidence for the role of La in poliovirus and HCV translation in vivo is lacking. Here, by two methods using small interfering RNA and a dominant-negative mutant of La, we demonstrate that depletion of La causes a dramatic reduction in poliovirus IRES function in vivo. We also show that 40S ribosomal subunit binding to HCV and poliovirus IRESs in vitro is inhibited by a dominant-negative form of La. These results provide strong evidence for a function of the La autoantigen in IRES-dependent translation and define the step of translation which is stimulated by La.

Internal ribosome entry site (IRES) from Encephalomyocarditis virus (EMCV) as a tool for shuttle expression plasmids

2015 ◽  
Vol 468 (4) ◽  
pp. 548-553 ◽  
Author(s):  
Sandra Aurora Telpalo-Carpio ◽  
Francisco Diaz-Mitoma ◽  
Jorge Eugenio Moreno-Cuevas ◽  
José Manuel Aguilar-Yáñez
Keyword(s):  
Internal Ribosome Entry Site ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Expression Plasmids

scholarly journals Internal Ribosome Entry Site Dramatically Reduces Transgene Expression in Hematopoietic Cells in a Position-Dependent Manner

Viruses ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 920 ◽  
Author(s):  
Qingyun Zheng ◽  
Xueyan Zhang ◽  
Hua Yang ◽  
Jinyan Xie ◽  
Yilin Xie ◽  
...  
Keyword(s):  
Internal Ribosome Entry Site ◽  
Transgene Expression ◽  
Hematopoietic Cells ◽  
Inhibitory Effect ◽  
Encephalomyocarditis Virus ◽  
Transduction Efficiency ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Virus Serotype

Bicistronic transgene expression mediated by internal ribosome entry site (IRES) elements has been widely used. It co-expresses heterologous transgene products from a message RNA driven by a single promoter. Hematologic gene delivery is a promising treatment for both inherited and acquired diseases. A combined strategy was recently documented for potential genome editing in hematopoietic cells. A transduction efficiency exceeding ~90% can be achieved by capsid-optimized recombinant adeno-associated virus serotype 6 (rAAV6) vectors. In this study, to deliver an encephalomyocarditis virus (EMCV) IRES-containing rAAV6 genome into hematopoietic cells, we observed that EMCV IRES almost completely shut down the transgene expression during the process of mRNA–protein transition. In addition, position-dependent behavior was observed, in which only the EMCV IRES element located between a promoter and the transgenes had an inhibitory effect. Although further studies are warranted to evaluate the involvement of cellular translation machinery, our results propose the use of specific IRES elements or an alternative strategy, such as the 2A system, to achieve bicistronic transgene expression in hematopoietic cells.

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