A selection system for functional internal ribosome entry site (IRES) elements: Analysis of the requirement for a conserved GNRA tetraloop in the encephalomyocarditis virus IRES

Robertson, Morwenna E. M.; Seamons, Rachael; Belsham, Graham J.

doi:10.1017/s1355838299990301

Cited by 85 publications

(84 citation statements)

References 26 publications

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“…The EMCV cDNA used was about 550 bp in length (nt 280-830 of the EMCV genome). The IRES mutant GCGC contains a point mutation within the highly conserved GNRA tetraloop within domain I, which strongly inhibits its activity (see Roberts and Belsham 1997;Robertson et al 1999). The DJ mutant has the domain J sequence (nt 696-730) precisely deleted, which also destroys the activity of the IRES when assayed within the context of a dicistronic reporter construct.…”

Section: Resultsmentioning

confidence: 99%

Monocistronic mRNAs containing defective hepatitis C virus-like picornavirus internal ribosome entry site elements in their 5′ untranslated regions are efficiently translated in cells by a cap-dependent mechanism

Belsham¹,

Nielsen²,

Normann³

et al. 2008

RNA

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The initiation of protein synthesis on mRNAs within eukaryotic cells is achieved either by a 59 cap-dependent mechanism or through internal initiation directed by an internal ribosome entry site (IRES). Picornavirus IRES elements, located in the 59 untranslated region (59UTR), contain extensive secondary structure and multiple upstream AUG codons. These features can be expected to inhibit cap-dependent initiation of translation. However, we have now shown that certain mutant hepatitis C virus-like picornavirus IRES elements (from porcine teschovirus-1 and avian encephalomyelitis virus), which are unable to direct internal initiation, are not significant barriers to efficient translation of capped monocistronic mRNAs that contain these defective elements within their 59UTRs. Moreover, the translation of these mRNAs is highly sensitive to the expression of an enterovirus 2A protease (which induces cleavage of eIF4G) and is also inhibited by hippuristanol, a specific inhibitor of eIF4A function, in contrast to their parental wild-type IRES elements. These results provide a possible basis for the evolution of viral IRES elements within the context of functional mRNAs that are translated by a cap-dependent mechanism.

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Section: Resultsmentioning

confidence: 99%

Monocistronic mRNAs containing defective hepatitis C virus-like picornavirus internal ribosome entry site elements in their 5′ untranslated regions are efficiently translated in cells by a cap-dependent mechanism

Belsham¹,

Nielsen²,

Normann³

et al. 2008

RNA

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“…These domains include highly specific binding sites for canonical initiation factors, auxiliary mRNA-binding proteins, or the 40 S ribosomal subunit (4). That is why short deletions or even point mutations in many parts of these IRESs are able to completely destroy them (21,(25)(26)(27)(28)(29)(30)(31)(32). This does not appear to be the case for the IRES of Hsp70 mRNA.…”

Section: Discussionmentioning

confidence: 99%

“…However, neither of these deleterious deletions abrogated completely the IRES activity of Hsp70 mRNA. This contrasts with the effect of small deletions or even point mutations on viral IRESs, at least those characterized to data (21,(25)(26)(27)(28)(29)(30)(31)(32). Some of such mutations completely abolish their translation initiation activity (see, for example, the data for one of the EMCV IRES mutants in Fig.…”

Section: Ires Properties Of the 5ј-utr Of Hsp70 Mrna Are Not Due To Ementioning

confidence: 97%

Distinctive Properties of the 5′-Untranslated Region of Human Hsp70 mRNA

Rubtsova

Sizova

Dmitriev

et al. 2003

Journal of Biological Chemistry

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A relaxed cap-dependence of translation of the mRNA-encoding mammalian heat shock protein Hsp70 may suggest that its 5 -untranslated region (UTR) possesses an internal ribosome entry site (IRES). In this study, this possibility has been tested in transfected cells using plasmids that express dicistronic mRNAs. Using a reporter gene construct, Renilla luciferase/Photinus pyralis luciferase, we show that the 216-nt long 5 -UTR of Hsp70 mRNA acts as an IRES that directs ribosomes to the downstream start codon by a cap-independent mechanism. The relative activity of this IRES (100-fold over the empty vector) is similar to that of the classical picornaviral IRESs. Additional controls indicate that this high expression of the downstream reporter is not due to readthrough from the upstream cistron, nor is it due to translation of cryptic monocistronic transcripts. The effect of small deletions within the 5 -UTR of Hsp70 mRNA on the IRES activity varies in dependence on their position within the 5 -UTR sequence. With the exception of deletion of nt 33-50, it is small for the 5 -terminal half of the 5 -UTR and rather strong for the 3 -terminal section. However, neither of these small deletions abolishes the IRES activity completely. Excision of larger sections (>50 nt) by truncation of the 5 -UTR from the 5 -end or by internal deleting results in a dramatic impairment of the IRES function. Taken together, these data suggest that the IRES activity of the 5 -UTR of Hsp70 mRNA requires integrity of almost the entire sequence of the 5 -UTR. The data are discussed in terms of a model that allows a three-dimensional rather than linear mode of selection of the initiation region surrounding the start codon of Hsp70 mRNA.

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“…Interestingly, the GNRA motif and the C-rich loop are conserved in IRES elements classified as type I or II (Fernandez et al, 2011a;Yu et al, 2011b). The GNRA motif, which is essential for IRES activity in both FMDV and EMCV (Lopez de Quinto and Martinez-Salas, 1997; Robertson et al, 1999), adopts a tetraloop conformation (Fernandez-Miragall and Martinez-Salas, 2003;Phelan et al, 2004), and determines long-range interactions (Fernandez-Miragall et al, 2006;Ramos and Martinez-Salas, 1999). Domain 4 consists of a Yshape RNA structure (corresponding to subdomains J and K) and provides the binding-site for eIF4G (Bassili et al, 2004;Kolupaeva et al, 1998;Lopez de Quinto and Martinez-Salas, 2000;Lozano et al, 2014).…”

Section: Picornavirus Ires Elements Diversitymentioning

confidence: 99%

Picornavirus IRES elements: RNA structure and host protein interactions

et al. 2015

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A selection system for functional internal ribosome entry site (IRES) elements: Analysis of the requirement for a conserved GNRA tetraloop in the encephalomyocarditis virus IRES

Abstract: Picornavirus internal ribosome entry site (IRES) elements direct cap-independent internal initiation

Cited by 85 publications

References 26 publications

Monocistronic mRNAs containing defective hepatitis C virus-like picornavirus internal ribosome entry site elements in their 5′ untranslated regions are efficiently translated in cells by a cap-dependent mechanism

Monocistronic mRNAs containing defective hepatitis C virus-like picornavirus internal ribosome entry site elements in their 5′ untranslated regions are efficiently translated in cells by a cap-dependent mechanism

Distinctive Properties of the 5′-Untranslated Region of Human Hsp70 mRNA

Picornavirus IRES elements: RNA structure and host protein interactions

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