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At least 19 recordsLinked to original sources
PubMed1999

Structure and function of a small RNA that selectively inhibits internal ribosome entry site-mediated translation.

A 60 nt long RNA termed IRNA, isolated from the yeast Saccharomyces cerevesiae, was previously shown to selectively block internal ribosome entry site (IRES)-mediated translation without interfering with cap-dependent translation of cellular mRNAs both in vivo and in vitro. IRNA specifically bound cellular proteins believed to be important for IRES-mediated translation. We demonstrate here that a complementary copy of IRNA (cIRNA) is also active in blocking IRES-mediated translation and that it binds many of the same cellular proteins that IRNA does. We have probed the secondary structure of both IRNA and cIRNA using single-strand- and double-strand-specific nucleases as well as using oligonucleotide hybridization followed by RNase H digestion. Both IRNA and cIRNA share secondary structural homology, although distinct differences do exist between the two structures. Mutational analysis of IRNA shows that sequences that form both the main stem and one loop are critical for its translation inhibitory activity. Maintenance of the established secondary structure appears to be required for both IRNA's ability to bind cellular trans -acting proteins believed to be required for IRES-mediated translation and its ability to block IRES-mediated translation.

Computer Simulation↗
PubMed2000

Identification of eIF2Bgamma and eIF2gamma as cofactors of hepatitis C virus internal ribosome entry site-mediated translation using a functional genomics approach.

The 5'-untranslated region of hepatitis C virus (HCV) is highly conserved, folds into a complex secondary structure, and functions as an internal ribosome entry site (IRES) to initiate translation of HCV proteins. We have developed a selection system based on a randomized hairpin ribozyme gene library to identify cellular factors involved in HCV IRES function. A retroviral vector ribozyme library with randomized target recognition sequences was introduced into HeLa cells, stably expressing a bicistronic construct encoding the hygromycin B phosphotransferase gene and the herpes simplex virus thymidine kinase gene (HSV-tk). Translation of the HSV-tk gene was mediated by the HCV IRES. Cells expressing ribozymes that inhibit HCV IRES-mediated translation of HSV-tk were selected via their resistance to both ganciclovir and hygromycin B. Two ribozymes reproducibly conferred the ganciclovir-resistant phenotype and were shown to inhibit IRES-mediated translation of HCV core protein but did not inhibit cap-dependent protein translation or cell growth. The functional targets of these ribozymes were identified as the gamma subunits of human eukaryotic initiation factors 2B (eIF2Bgamma) and 2 (eIF2gamma), respectively. The involvement of eIF2Bgamma and eIF2gamma in HCV IRES-mediated translation was further validated by ribozymes directed against additional sites within the mRNAs of these genes. In addition to leading to the identification of cellular IRES cofactors, ribozymes obtained from this cellular selection system could be directly used to specifically inhibit HCV viral translation, thereby facilitating the development of new antiviral strategies for HCV infection.

5' Untranslated Regions↗
PubMed2000

Human La antigen is required for the hepatitis C virus internal ribosome entry site-mediated translation.

The 5'-noncoding region (5'-NCR) of the hepatitis C virus (HCV) RNA genome serves as an internal ribosome entry site (IRES) and mediates translation initiation in a cap-independent manner. Previously, we reported the interaction between La antigen and the HCV IRES, which appeared to occur in the context of initiator AUG. It was further shown that HCV IRES-mediated translation was stimulated in the presence of human La antigen. In this study, we have defined the cis- and trans-acting elements responsible for La-5'-NCR interactions and established the dependence of the HCV IRES efficiency on cellular La antigen. During the La-IRES interaction, initiator AUG but not the neighboring codons was found to be the direct target of La binding. The C terminus effector domain-dependent modulation of La binding to the HCV IRES is demonstrated by deletion and substitution mutagenesis of the protein. An RNA systematic evolution of ligands by exponential enrichment (SELEX), generated against La protein that selectively binds La in HeLa lysates and competes for the protein binding to the 5'-NCR, was used to demonstrate the requirement of La for the HCV IRES function in the context of mono- and dicistronic mRNAs. Sequestration of La antigen by the RNA SELEX in HeLa translation lysates blocked the HCV and poliovirus IRES-mediated translation in vitro. The functional requirement of La protein for the HCV IRES activity was further established in a liver-derived cell line and in an add-back experiment in which the inhibited IRES was rescued by recombinant human La. These results strongly argue for the novel role of La protein during selection of the initiator AUG and its participation during internal initiation of translation of the HCV RNA genome.

5' Untranslated Regions↗
PubMed2000

Transcription-coupled translation control of AML1/RUNX1 is mediated by cap- and internal ribosome entry site-dependent mechanisms.

AML1/RUNX1 belongs to the runt domain transcription factors that are important regulators of hematopoiesis and osteogenesis. Expression of AML1 is regulated at the level of transcription by two promoters, distal (D) and proximal (P), that give rise to mRNAs bearing two distinct 5' untranslated regions (5'UTRs) (D-UTR and P-UTR). Here we show that these 5'UTRs act as translation regulators in vivo. AML1 mRNAs bearing the uncommonly long (1,631-bp) P-UTR are poorly translated, whereas those with the shorter (452-bp) D-UTR are readily translated. The low translational efficiency of the P-UTR is attributed to its length and the cis-acting elements along it. Transfections and in vitro assays with bicistronic constructs demonstrate that the D-UTR mediates cap-dependent translation whereas the P-UTR mediates cap-independent translation and contains a functional internal ribosome entry site (IRES). The IRES-containing bicistronic constructs are more active in hematopoietic cell lines that normally express the P-UTR-containing mRNAs. Furthermore, we show that the IRES-dependent translation increases during megakaryocytic differentiation but not during erythroid differentiation, of K562 cells. These results strongly suggest that the function of the P-UTR IRES-dependent translation in vivo is to tightly regulate the translation of AML1 mRNAs. The data show that AML1 expression is regulated through usage of alternative promoters coupled with IRES-mediated translation control. This IRES-mediated translation regulation adds an important new dimension to the fine-tuned control of AML1 expression.

5' Untranslated Regions↗
PubMed1998

Inhibition of internal entry site (IRES)-mediated translation by a small yeast RNA: a novel strategy to block hepatitis C virus protein synthesis.

The observation that poliovirus mRNA is not translated in the yeast Saccharomyces cerevisiae has led to the discovery of a small RNA (60 nt, called IRNA, inhibitor RNA) which was later shown to specifically inhibit internal ribosome entry site (IRES)-mediated translation of naturally uncapped mRNAs. Translation of cellular capped mRNAs was not significantly inhibited by IRNA. IRNA also specifically inhibited hepatitis C virus (HCV) IRES-mediated translation in vitro and in vivo. A hepatoma cell line constitutively expressing IRNA was refractory to infection by a chimeric poliovirus (PV/HCV) in which PV IRES is replaced by HCV-IRES. In contrast, a PV/EMCV chimeric virus containing the EMCV IRES was not significantly inhibited in the IRNA-hepatoma cell line compared to the control hepatoma cells. UV-crosslinking studies showed that the IRNA binds a number of cellular proteins that appear to be important for IRES-mediated translation. Interaction of these proteins with the viral IRES elements is believed to be important in recruiting ribosomes to the 5( UTR of viral RNAs. The binding of the purified La autoantigen to the HCV IRES element was efficiently and specifically competed by IRNA. These results provide a basis for development of novel drugs effective against HCV infection.

Animals↗
PubMed2000

c-Myc protein synthesis is initiated from the internal ribosome entry segment during apoptosis.

Recent studies have shown that during apoptosis protein synthesis is inhibited and that this is in part due to the proteolytic cleavage of eukaryotic initiation factor 4G (eIF4G). Initiation of translation can occur either by a cap-dependent mechanism or by internal ribosome entry. The latter mechanism is dependent on a complex structural element located in the 5' untranslated region of the mRNA which is termed an internal ribosome entry segment (IRES). In general, IRES-mediated translation does not require eIF4E or full-length eIF4G. In order to investigate whether cap-dependent and cap-independent translation are reduced during apoptosis, we examined the expression of c-Myc during this process, since we have shown previously that the 5' untranslated region of the c-myc proto-oncogene contains an IRES. c-Myc expression was determined in HeLa cells during apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand. We have demonstrated that the c-Myc protein is still expressed when more than 90% of the cells are apoptotic. The presence of the protein in apoptotic cells does not result from either an increase in protein stability or an increase in expression of c-myc mRNA. Furthermore, we show that during apoptosis initiation of c-myc translation occurs by internal ribosome entry. We have investigated the signaling pathways that are involved in this response, and cotransfection with plasmids which harbor either wild-type or constitutively active MKK6, a specific immediate upstream activator of p38 mitogen-activated protein kinase (MAPK), increases IRES-mediated translation. In addition, the c-myc IRES is inhibited by SB203580, a specific inhibitor of p38 MAPK. Our data, therefore, strongly suggest that the initiation of translation via the c-myc IRES during apoptosis is mediated by the p38 MAPK pathway.

Apoptosis↗
PubMed1998

A small yeast RNA blocks hepatitis C virus internal ribosome entry site (HCV IRES)-mediated translation and inhibits replication of a chimeric poliovirus under translational control of the HCV IRES element.

Hepatitis C virus (HCV) infection frequently leads to chronic hepatitis and cirrhosis of the liver and has been linked to development of hepatocellular carcinoma. We previously identified a small yeast RNA (IRNA) capable of specifically inhibiting poliovirus (PV) internal ribosome entry site (IRES)-mediated translation. Here we report that IRNA specifically inhibits HCV IRES-mediated translation both in vivo and in vitro. A number of human hepatoma (Huh-7) cell lines expressing IRNA were prepared and characterized. Constitutive expression of IRNA was not detrimental to cell growth. HCV IRES-mediated cap-independent translation was markedly inhibited in cells constitutively expressing IRNA compared to control hepatoma cells. However, cap-dependent translation was not significantly affected in these cell lines. Additionally, Huh-7 cells constitutively expressing IRNA became refractory to infection by a PV-HCV chimera in which the PV IRES is replaced by the HCV IRES. In contrast, replication of a PV-encephalomyocarditis virus (EMCV) chimera containing the EMCV IRES element was not affected significantly in the IRNA-producing cell line. Finally, the binding of the La autoantigen to the HCV IRES element was specifically and efficiently competed by IRNA. These results provide a basis for development of novel drugs effective against HCV infection.

Autoantigens↗
PubMed1996

Sequences within a small yeast RNA required for inhibition of internal initiation of translation: interaction with La and other cellular proteins influences its inhibitory activity.

We recently reported purification, determination of the nucleotide sequence, and cloning of a 60-nucleotide RNA (I-RNA) from the yeast Saccharomyces cerevisiae which preferentially blocked cap-independent, internal ribosome entry site (IRES)-mediated translation programmed by the poliovirus (PV) 5' untranslated region (UTR). The I-RNA appeared to inhibit IRES-mediated translation by virtue of its ability to bind a 52-kDa polypeptide which interacts with the 5' UTR of viral RNA. We demonstrate here that the HeLa 52-kDa I-RNA-binding protein is immunologically identical to human La autoantigen. Moreover, I-RNA-mediated purified La protein. By using I-RNAs with defined deletions, we have identified sequences of I-RNA required for inhibition of internal initiation of translation. Two smaller fragments of I-RNA (16 and 25 nucleotides) inhibited PV UTR-mediated translation from both monocistronic and bicistronic RNAs. When transfected into HeLa cells, these derivatives of I-RNA inhibited translation of PV RNA. A comparison of protein binding by active and inactive I-RNA mutants demonstrates that in addition to the La protein, three other polypeptides with apparent molecular masses of 80, 70, and 37 kDa may influence the translation-inhibitory activity of I-RNA.

Autoantigens↗
PubMed1997

Translation and replication properties of the human rhinovirus genome in vivo and in vitro.

The poor translation efficiency of genome-length human rhinovirus RNA in vitro using HeLa cell extract-supplemented rabbit reticulocyte lysate has hampered the study of rhinovirus IRES-mediated translation and polyprotein synthesis in a cell-free system. In contrast, the efficient in vitro translation characteristics of poliovirus RNAs have ultimately allowed the programming of cell-free coupled translation/replication extracts which are able to produce infectious poliovirus particles in vitro. A possible explanation for the decreased burst size observed during the course of a rhinovirus infection, compared to poliovirus infection, is reduced levels of polyprotein synthesis in vivo. In order to test this hypothesis and extend in vitro translation/replication technology to the study of human rhinoviruses, a chimeric cDNA construct was engineered which allowed the in vitro synthesis of T7 transcripts containing the intact poliovirus type 1 (PV1) 5' noncoding region (5' NCR) and initiation codon upstream of the human rhinovirus 14 (HRV14) polyprotein-coding region and 3'-terminal sequences. These chimeric RNAs translated efficiently in vitro and were used successfully to program a cell-free replication extract. Unexpectedly, parental HRV14 RNAs also translated efficiently in the HeLa cell-free translation/replication extract but replicated less efficiently than the chimera in vitro. The chimeric HRV14/PV1 RNAs were infectious and gave rise to a virus with a growth phenotype similar to that of parental HRV14. Preliminary characterization of this chimeric virus suggests that the biological properties characteristic of rhinovirus in vivo are determined primarily by the rhinovirus gene products. Although the translation efficiency of the HRV14 5' NCR may be a limitation in rabbit reticulocyte lysate-based in vitro translation extracts, it does not appear to be a major limiting determinant for growth of rhinovirus in vivo or replication in the HeLa cell-free extract.

Animals↗
PubMed1999

Lower concentration of La protein required for internal ribosome entry on hepatitis C virus RNA than on poliovirus RNA.

Translation initiation of poliovirus and hepatitis C virus (HCV) RNA occurs by entry of ribosomes to the internal RNA sequence, called the internal ribosomal entry site (IRES). Both IRES bind to the La protein and are thought to require the protein for their translation initiation activity, although they are greatly different in both the primary and predicted secondary structures. To compare the La protein requirement for these IRES, we took advantage of I-RNA from the yeast Saccharomyces cerevisiae, which has been reported to bind to La protein and block poliovirus IRES-mediated translation initiation. In a cell-free translation system prepared from HeLa cells, yeast I-RNA inhibited translation initiation on poliovirus RNA as expected, but did not significantly inhibit translation initiation on HCV RNA. However, the translation initiation directed by either IRES was apparently inhibited by I-RNA in rabbit reticulocyte lysates, in which La protein is limiting. I-RNA-mediated inhibition of HCV IRES-dependent translation in rabbit reticulocyte lysates was reversed by exogenous addition of purified recombinant La protein of smaller amounts than necessary to reverse poliovirus IRES-dependent translation. These results suggest that HCV IRES requires lower concentrations of La protein for its function than does poliovirus IRES. Immunofluorescence studies showed that HCV infection appeared not to affect the subcellular localization of La protein, which exists mainly in the nucleus, although La protein redistributed to the cytoplasm after poliovirus infection. The data are compatible with the low requirement of La protein for HCV IRES activity.

Animals↗
PubMed1997

Internal entry of ribosomes is directed by the 5' noncoding region of classical swine fever virus and is dependent on the presence of an RNA pseudoknot upstream of the initiation codon.

Bicistronic RNAs containing the 373-nucleotide-long 5' nontranslated region (NTR) of the classical swine fever virus (CSFV) genome as intercistronic spacer were used to show the presence of an internal ribosome entry site (IRES) in the 5' end of the CSFV genome. By coexpression of the poliovirus 2A protease it was demonstrated that the CSFV 5' NTR-driven translation is independent of the presence of functional eukaryotic initiation factor eIF-4F. Deletion analysis indicated that the 5' border of the IRES is located between nucleotides 28 and 66. The role of a proposed pseudoknot structure at the 3' end of the CSFV 5' NTR in IRES-mediated translation was investigated by site-directed mutagenesis. Mutant RNAs that had lost the ability to base pair in stem II of the pseudoknot were translationally inactive. Translation to wild-type levels could be restored through the introduction of compensatory complementary base changes that repaired base pairing in stem II. In addition, we showed that the AUG codon, which is located 7 nucleotides upstream of the polyprotein initiation site and is conserved in pestiviruses, could not be used to initiate translation. Also, an AUG codon introduced downstream of the polyprotein initiation site was not recognized as an initiation site by ribosomes. These data suggest that after internal entry on the CSFV 5' NTR, ribosomal scanning for the initiation codon is limited to a small region.

Animals↗
PubMed1999

Translation initiation at the CUU codon is mediated by the internal ribosome entry site of an insect picorna-like virus in vitro.

AUG-unrelated translation initiation was found in an insect picorna-like virus, Plautia stali intestine virus (PSIV). The positive-strand RNA genome of the virus contains two nonoverlapping open reading frames (ORFs). The capsid protein gene is located in the 3'-proximal ORF and lacks an AUG initiation codon. We examined the translation mechanism and the initiation codon of the capsid protein gene by using various dicistronic and monocistronic RNAs in vitro. The capsid protein gene was translated cap independently in the presence of the upstream cistron, indicating that the gene is translated by internal ribosome entry. Deletion analysis showed that the internal ribosome entry site (IRES) consisted of approximately 250 bases and that its 3' boundary extended slightly into the capsid-coding region. The initiation codon for the IRES-mediated translation was identified as the CUU codon, which is located just upstream of the 5' terminus of the capsid-coding region by site-directed mutagenesis. In vitro translation assays of monocistronic RNAs lacking the 5' part of the IRES showed that this CUU codon was not recognized by scanning ribosomes. This suggests that the PSIV IRES can effectively direct translation initiation without stable codon-anticodon pairing between the initiation codon and the initiator methionyl-tRNA.

Amino Acid Sequence↗
PubMed2000

Mutational analysis of the GB virus B internal ribosome entry site.

GB virus B (GBV-B) is a recently discovered hepatotropic flavivirus that is distantly related to hepatitis C virus (HCV). We show here that translation of its polyprotein is initiated by internal entry of ribosomes on GBV-B RNA. We analyzed the translational activity of dicistronic RNA transcripts containing wild-type or mutated 5' nontranslated GBV-B RNA (5'NTR) segments, placed between the coding sequences of two reporter proteins, in vitro in rabbit reticulocyte lysate and in vivo in transfected BT7-H cells. We related these results to a previously proposed model of the secondary structure of the GBV-B 5'NTR (M. Honda, et al. RNA 2:955-968, 1996). We identified an internal ribosome entry site (IRES) bounded at its 5' end by structural domain II, a location analogous to the 5' limit of the IRES in both the HCV and pestivirus 5'NTRs. Mutational analysis confirmed the structure proposed for domain II of GBV-B RNA, and demonstrated that optimal IRES-mediated translation is dependent on each of the putative RNA hairpins in this domain, including two stem-loops not present in the HCV or pestivirus structures. IRES activity was also absolutely dependent on (i) phylogenetically conserved, adenosine-containing bulge loops in domain III and (ii) the primary nucleotide sequence of stem-loop IIIe. IRES-directed translation was inhibited by a series of point mutations predicted to stabilize stem-loop IV, which contains the initiator AUG codon in its loop segment. A reporter gene was translated most efficiently when fused directly to the initiator AUG codon, with no intervening downstream GBV-B sequence. This finding indicates that the 3' limit of the GBV-B IRES is at the initiator AUG and that it does not require downstream polyprotein-coding sequence as suggested for the HCV IRES. These results show that the GBV-B IRES, while sharing a common general structure, differs both structurally and functionally from other flavivirus IRES elements.

5' Untranslated Regions↗
PubMed2000

Functional characterization of the X-linked inhibitor of apoptosis (XIAP) internal ribosome entry site element: role of La autoantigen in XIAP translation.

X-linked inhibitor of apoptosis protein (XIAP) is a key regulator of programmed cell death triggered by various apoptotic triggers. Translation of XIAP is controlled by a 162-nucleotide (nt) internal ribosome entry site (IRES) element located in the 5' untranslated region of XIAP mRNA. XIAP IRES mediates efficient translation of XIAP under physiological stress and enhances cell protection against serum deprivation and radiation-induced apoptosis. In the present report we describe the assembly of a sequence-specific RNA-protein complex consisting of at least four cytosolic proteins on the XIAP IRES element. We determine that the core binding sequence is approximately 28 nt long and is located 34 nt upstream of the initiation site. Moreover, we identify the La autoantigen as a protein that specifically binds XIAP IRES in vivo and in vitro. The biological relevance of this interaction is further demonstrated by the inhibition of XIAP IRES-mediated translation in the absence of functional La protein. The results suggest an important role for the La protein in the regulation of XIAP expression, possibly by facilitating ribosome recruitment to the XIAP IRES.

Autoantigens↗
PubMed2026

PTBP1 at the host-virus interface: mechanistic roles in viral RNA translation, replication, and immune modulation.

Viruses require the involvement of host RNA binding proteins for completion of important steps of their life cycle. Polypyrimidine tract binding protein 1 (PTBP1) is an RNA-binding protein found ubiquitously which performs important regulatory functions like alternative splicing, RNA stability, RNA localization, and translation by virtue of its four RRMs and shuttling between nucleus and cytoplasm. There is increasing evidence showing that many viruses make use of such regulatory roles of PTBP1 to facilitate their gene expression and replication. This review describes the existing mechanistic knowledge about the PTBP1 functions during viral infection, paying attention to the role of PTBP1 in viral RNA translation, viral RNA genome replication, and regulation of host antiviral response. Special attention is paid to the regulation by PTBP1 of IRES-dependent translation of enteroviruses and hepatitis C virus, as well as to the PTBP1 contribution to RNA stabilization, long-distance RNA interactions, and genome cyclization of flaviviruses such as dengue virus and Japanese encephalitis virus. Recent data on the PTBP1 function in coronavirus RNA metabolism are discussed as well. Furthermore, the role of PTBP1 in being both proviral and antiviral is reviewed in terms of innate immunity signalling pathways, stress granule biology, and virus-host interaction. Finally, we will explore the possibility of PTBP1 being used as a host-directed antiviral drug target despite the hurdles in doing so considering its multifunctionality as an essential cellular RNA-binding protein.

Polypyrimidine Tract-Binding Protein↗
PubMed1996

Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes.

Eukaryotic translation is initiated following binding of ribosomes either to the capped 5' end of an mRNA or to an internal ribosomal entry site (IRES) within its 5' nontranslated region. These processes are both mediated by eukaryotic initiation factor 4F (eIF4F), which consists of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G subunits. Here we present a functional analysis of eIF4F which defines the subunits and subunit domains necessary for its function in initiation mediated by the prototypical IRES element of encephalomyocarditis virus. In an initiation reaction reconstituted in vitro from purified translation components and lacking eIF4A and -4F, IRES-mediated initiation did not require the cap-binding protein eIF4E but was absolutely dependent on eIF4A and the central third of eIF4G. This central domain of eIF4G bound strongly and specifically to a structural element within the encephalomyocarditis virus IRES upstream of the initiation codon in an ATP-independent manner and with the same specificity as eIF4F. The carboxy-terminal third of eIF4G did not bind to the IRES. The central domain of eIF4G was itself UV cross-linked to the IRES and strongly stimulated UV cross-linking of eIF4A to the IRES in conjunction with either eIF4B or with the carboxy-terminal third of eIF4G.

Encephalomyocarditis virus↗
PubMed1992

The sequence context of the initiation codon in the encephalomyocarditis virus leader modulates efficiency of internal translation initiation.

Translation initiation on poliovirus and encephalomyocarditis virus (EMCV) mRNAs occurs by a cap-independent mechanism utilizing an internal ribosomal entry site (IRES). However, no unifying mechanism for AUG initiation site selection has been proposed. Analysis of initiation of mRNAs translated in vitro has suggested that initiation of poliovirus mRNA translation likely involves both internal binding of ribosomes and scanning to the first AUG which is in a favorable context for initiation. In contrast, internal initiation on EMCV mRNA may not utilize scanning, since ribosomes bind directly or very close to the initiation codon AUG-11. We have studied in vivo the sequence requirements for internal initiation around the EMCV initiation codon, both in monocistronic and in dicistronic mRNAs. Our studies show that the upstream AUG-10 is normally not used and that there is no specific sequence requirement for nucleotides between AUG-10 and AUG-11. However, the sequence context of AUG-11 does influence the efficiency of initiation at AUG-11. Efficient IRES-mediated internal initiation at AUG-11 exhibits a requirement for an adenine in the -3 position, similar to cap-dependent initiation. These results support a model for internal initiation on EMCV mRNA in which scanning starts at or near AUG-11. Although initiation primarily occurs at AUG-11, initiation at multiple downstream AUG codons can be detected. In addition, a poor sequence context around AUG-11 results in increased initiation at one or more downstream AUG codons, indicative of leaky scanning or jumping by the ribosome from AUG-11 mediated by the EMCV IRES.

Animals↗
PubMed1996

Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry.

Translation of picornavirus RNA is initiated after ribosomal binding to an internal ribosomal entry site (IRES) within the 5' untranslated region. We have reconstituted IRES-mediated initiation on encephalomyocarditis virus RNA from purified components and used primer extension analysis to confirm the fidelity of 48S preinitiation complex formation. Eukaryotic initiation factor 2 (eIF2), eIF3, and eIF4F were required for initiation; eIF4B and to a lesser extent the pyrimidine tract-binding protein stimulated this process. We show that eIF4F binds to the IRES in a novel cap-independent manner and suggest that cap- and IRES-dependent initiation mechanisms utilize different modes of interaction with this factor to promote ribosomal attachment to mRNA.

Base Sequence↗