scholarly journals Construction of a bifunctional mRNA in the mouse by using the internal ribosomal entry site of the encephalomyocarditis virus.

1992 ◽  
Vol 12 (8) ◽  
pp. 3636-3643 ◽  
Author(s):  
D G Kim ◽  
H M Kang ◽  
S K Jang ◽  
H S Shin
Keyword(s):  
Es Cells ◽  
Internal Ribosomal Entry Site ◽  
Embryonic Stem ◽  
Mouse Embryos ◽  
Encephalomyocarditis Virus ◽  
Vector System ◽  
Chimeric Mouse ◽  
Entry Site ◽  
Cell Extracts ◽  
Ribosomal Entry

Picornaviral mRNAs have been shown to possess special structures in their 5' nontranslated regions (5'NTRs) that provide sites for internal binding of ribosomes and thus direct cap-independent translation. The translational cis-acting elements for ribosomal internal entry into the 5'NTR of encephalomyocarditis virus (EMCV), a member of family Picornaviridae, have been named the internal ribosomal entry site (IRES). All of the published experiments regarding the IRES function of the picornavirus 5'NTR, however, were performed with cell extracts in vitro or with tissue culture cells in transient assay systems. In this study, we examined the IRES function of the EMCV 5'NTR in chimeric mouse embryos and demonstrated that this element does in fact work stably in mouse embryos as well as in embryonic stem (ES) cells. By using a dicistronic vector, pWH8, consisting of a promoter-driven neomycin resistance gene (neo) followed by the EMCV 5'NTR-lacZ sequence, we showed that more than half of the ES cells made G418 resistant by the vector stained positive for beta-galactosidase (beta-gal). On Northern (RNA) blots, all of the clones analyzed revealed a transcript of the expected size containing both the beta-gal and the neo cistrons. These results indicate that dicistronic mRNAs are produced from the stably integrated vector in those ES clones and that both of the cistrons are translated to produce functional proteins. The chimeric embryos derived from these ES clones also stained positive for beta-gal, suggesting that the bifunctional mRNAs are active in the embryos. This dicistronic vector system provides a novel tool by which to obtain temporally and spatially coordinated expression of two different genes driven by a single promoter in a single cell in mice.

Construction of a bifunctional mRNA in the mouse by using the internal ribosomal entry site of the encephalomyocarditis virus

1992 ◽  
Vol 12 (8) ◽  
pp. 3636-3643
Author(s):  
D G Kim ◽  
H M Kang ◽  
S K Jang ◽  
H S Shin
Keyword(s):  
Es Cells ◽  
Internal Ribosomal Entry Site ◽  
Embryonic Stem ◽  
Mouse Embryos ◽  
Encephalomyocarditis Virus ◽  
Vector System ◽  
Chimeric Mouse ◽  
Entry Site ◽  
Cell Extracts ◽  
Ribosomal Entry

Picornaviral mRNAs have been shown to possess special structures in their 5' nontranslated regions (5'NTRs) that provide sites for internal binding of ribosomes and thus direct cap-independent translation. The translational cis-acting elements for ribosomal internal entry into the 5'NTR of encephalomyocarditis virus (EMCV), a member of family Picornaviridae, have been named the internal ribosomal entry site (IRES). All of the published experiments regarding the IRES function of the picornavirus 5'NTR, however, were performed with cell extracts in vitro or with tissue culture cells in transient assay systems. In this study, we examined the IRES function of the EMCV 5'NTR in chimeric mouse embryos and demonstrated that this element does in fact work stably in mouse embryos as well as in embryonic stem (ES) cells. By using a dicistronic vector, pWH8, consisting of a promoter-driven neomycin resistance gene (neo) followed by the EMCV 5'NTR-lacZ sequence, we showed that more than half of the ES cells made G418 resistant by the vector stained positive for beta-galactosidase (beta-gal). On Northern (RNA) blots, all of the clones analyzed revealed a transcript of the expected size containing both the beta-gal and the neo cistrons. These results indicate that dicistronic mRNAs are produced from the stably integrated vector in those ES clones and that both of the cistrons are translated to produce functional proteins. The chimeric embryos derived from these ES clones also stained positive for beta-gal, suggesting that the bifunctional mRNAs are active in the embryos. This dicistronic vector system provides a novel tool by which to obtain temporally and spatially coordinated expression of two different genes driven by a single promoter in a single cell in mice.

scholarly journals Unr Is Required In Vivo for Efficient Initiation of Translation from the Internal Ribosome Entry Sites of both Rhinovirus and Poliovirus

2003 ◽  
Vol 77 (6) ◽  
pp. 3353-3359 ◽  
Author(s):  
Oréda Boussadia ◽  
Michael Niepmann ◽  
Laurent Créancier ◽  
Anne-Catherine Prats ◽  
François Dautry ◽  
...  
Keyword(s):  
Rna Binding ◽  
Es Cells ◽  
Internal Ribosomal Entry Site ◽  
Embryonic Stem ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Initiation Of Translation ◽  
Mouth Disease Virus

ABSTRACT Translation of picornavirus RNAs is mediated by internal ribosomal entry site (IRES) elements and requires both standard eukaryotic translation initiation factors (eIFs) and IRES-specific cellular trans-acting factors (ITAFs). Unr, a cytoplasmic RNA-binding protein that contains five cold-shock domains and is encoded by the gene upstream of N-ras, stimulates translation directed by the human rhinovirus (HRV) IRES in vitro. To examine the role of Unr in translation of picornavirus RNAs in vivo, we derived murine embryonic stem (ES) cells in which either one (−/+) or both (−/−) copies of the unr gene were disrupted by homologous recombination. The activity of picornaviral IRES elements was analyzed in unr +/+, unr +/−, and unr −/− cell lines. Translation directed by the HRV IRES was severely impaired in unr −/− cells, as was that directed by the poliovirus IRES, revealing a requirement for Unr not previously observed in vitro. Transient expression of Unr in unr −/− cells efficiently restored the HRV and poliovirus IRES activities. In contrast, the IRES elements of encephalomyocarditis virus and foot-and-mouth-disease virus are not Unr dependent. Thus, Unr is a specific regulator of HRV and poliovirus translation in vivo and may represent a cell-specific determinant limiting replication of these viruses.

scholarly journals Heat Shock Protein A6, a Novel HSP70, Is Induced During Enterovirus A71 Infection to Facilitate Internal Ribosomal Entry Site-Mediated Translation

2021 ◽  
Vol 12 ◽  
Author(s):  
Yu-Siang Su ◽  
Lih-Hwa Hwang ◽  
Chi-Ju Chen
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Neurological Diseases ◽  
Internal Ribosomal Entry Site ◽  
Viral Proteins ◽  
Cellular Protein ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Enterovirus A71 ◽  
Ribosomal Entry

Enterovirus A71 (EV-A71) is a human pathogen causing hand, foot, and mouth disease (HFMD) in children. Its infection can lead to severe neurological diseases or even death in some cases. While being produced in a large quantity during infection, viral proteins often require the assistance from cellular chaperones for proper folding. In this study, we found that heat shock protein A6 (HSPA6), whose function in viral life cycle is scarcely studied, was induced and functioned as a positive regulator for EV-A71 infection. Depletion of HSPA6 led to the reductions of EV-A71 viral proteins, viral RNA and virions as a result of the downregulation of internal ribosomal entry site (IRES)-mediated translation. Unlike other HSP70 isoforms such as HSPA1, HSPA8, and HSPA9, which regulate all phases of the EV-A71 life, HSPA6 was required for the IRES-mediated translation only. Unexpectedly, the importance of HSPA6 in the IRES activity could be observed in the absence of viral proteins, suggesting that HSPA6 facilitated IRES activity through cellular factor(s) instead of viral proteins. Intriguingly, the knockdown of HSPA6 also caused the reduction of luciferase activity driven by the IRES from coxsackievirus A16, echovirus 9, encephalomyocarditis virus, or hepatitis C virus, supporting that HSPA6 may assist the function of a cellular protein generally required for viral IRES activities.

scholarly journals cis-Acting Elements of the Encephalomyocarditis Virus Internal Ribosomal Entry Site

Virology ◽  
1995 ◽  
Vol 214 (2) ◽  
pp. 660-663 ◽  
Author(s):  
GARY W. WITHERELL ◽  
CARLA S. SCHULTZ-WITHERELL ◽  
ECKARD WIMMER
Keyword(s):  
Internal Ribosomal Entry Site ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Cis Acting ◽  
Ribosomal Entry

scholarly journals La protein is required for efficient translation driven by encephalomyocarditis virus internal ribosomal entry site

1999 ◽  
Vol 80 (12) ◽  
pp. 3159-3166 ◽  
Author(s):  
Yoon Ki Kim ◽  
Sung Key Jang
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein A ◽  
Internal Ribosomal Entry Site ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Polypyrimidine Tract Binding Protein ◽  
La Protein ◽  
Ribosomal Entry

Translation of internal ribosomal entry site (IRES)-dependent mRNAs is mediated by RNA-binding proteins as well as canonical translation factors. In order to elucidate the roles of RNA-binding proteins in IRES-dependent translation, the role of polypyrimidine tract-binding protein (PTB) and La protein in encephalomyocarditis virus (EMCV) IRES-dependent translation was investigated. PTB was required for efficient EMCV IRES-driven translation but, intriguingly, an excess of PTB suppressed it. Such a translational suppression by surplus PTB was relieved by addition of La protein. A possible role for La protein in IRES-dependent translation is discussed.

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