Starved Saccharomyces cerevisiae cells have the capacity to support internal initiation of translation.

Internal initiation of translation, whereby ribosomes are directed to internal AUG codon independently of the 5' end of the mRNA, has been observed rarely in higher eucaryotes and has not been demonstrated in living yeast. We report here that starved yeast cells are capable of initiating translation of a dicistronic message internally. The studied element that functions as an internal ribosome entry site (IRES) is hardly functional or not functional at all in logarithmically growing cells. Moreover, during the logarithmic growth phase, this element seems to inhibit translation reinitiation when placed as an intercistronic spacer or to inhibit translation when placed in the 5'-untranslated region of a monocistronic message. Inhibition of translation is likely due to the putative strong secondary structure of the IRES that interferes with the cap-dependent scanning process. When cells exit the logarithmic growth phase, or when artificially starved for carbon source, translation of the IRES-containing messages is substantially induced. Our findings imply that the capacity to translate internally is a characteristic of starved rather than vegetatively growing yeast cells.     

Cap-independent polysomal association of natural mRNAs encoding c-myc, BiP, and eIF4G conferred by internal ribosome entry sites.

Sequence elements that can function as internal ribosome entry sites (IRES) have been identified in 5' noncoding regions of certain uncapped viral and capped cellular mRNA molecules. However, it has remained largely unknown whether IRES elements are functional when located in their natural capped mRNAs. Therefore, the polysomal association and translation of several IRES-containing cellular mRNAs was tested under conditions that severely inhibited cap-dependent translation, that is, after infection with poliovirus. It was found that several known IRES-containing mRNAs, such as BiP and c-myc, were both associated with the translation apparatus and translated in infected cells when cap-dependent translation of most host-cell mRNAs was blocked, indicating that the IRES elements were functional in their natural mRNAs. Curiously, the mRNAs that encode eukaryotic initiation factor 4GI (eIF4GI) and 4GII (eIF4GII), two proteins with high identity and similar functions in the initiation of cap-dependent translation, were both associated with polysomes in infected cells. The 5'-end sequences of eIF4GI mRNA were isolated from a cDNA expression library and shown to function as an internal ribosome entry site when placed into a dicistronic mRNA. These findings suggest that eIF4G proteins can be synthesized at times when 5' cap-dependent mRNA translation is blocked, supporting the notion that eIF4G proteins are needed in both 5' cap-independent and 5' cap-dependent translational initiation mechanisms.     

Competitive translation efficiency at the picornavirus type 1 internal ribosome entry site facilitated by viral cis and trans factors

Enteroviruses (EVs) overcome their host cells by usurping the translation machinery to benefit viral gene expression. This is accomplished through alternative translation initiation in a cap-independent manner at the viral internal ribosomal entry site (IRES). We have investigated the role of cis- and trans-acting viral factors in EV IRES translation in living cells. We observed that considerable portions of the viral genome, including the 5'-proximal open reading frame and the 3' untranslated region, contribute to stimulation of IRES-mediated translation. With the IRES in proper context, translation via internal initiation in uninfected cells is as efficient as at capped messages with short, unstructured 5' untranslated regions. IRES function is enhanced in cells infected with the EV coxsackievirus B3, but the related poliovirus has no significant stimulatory activity. This differential is due to the inherent properties of their 2A protease and is not coupled to 2A-mediated proteolytic degradation of the eukaryotic initiation factor 4G. Our results suggest that the efficiency of alternative translation initiation at EV IRESs depends on a properly configured template rather than on targeted alterations of the host cell translation machinery.     

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.     

Role of GNRA motif mutations within stem-loop V of internal ribosome entry segment in coxsackievirus B3 molecular attenuation

The lengthy 5' nontranslated region of coxsackievirus B3 (CVB3) forms a highly ordered secondary structure containing an internal ribosome entry segment (IRES), which plays an important role in controlling viral translation and pathogenesis. The stem-loop V (SL-V) of this IRES contains a large lateral bulge loop which encompasses two conserved GNRA motifs. In this study, we analyzed the effects of point mutations within the GNRA motifs of the CVB3 IRES. We characterized in vitro virus production and translation efficiency and we tested in vivo virulence of two CVB3 mutants produced by site-directed mutagenesis. The GNAA1 and GNAA2 RNAs displayed decreased translation initiation efficiency when translated in rabbit reticulocyte lysates. This translation defect was correlated with reduced yields of infectious virus particles in HeLa cells in comparison with the wild type. When inoculated orally into Swiss mice, both mutant viruses were avirulent and caused neither inflammation nor necrosis in hearts. These results highlight the important role of the GNRA motifs within the SL-V of the IRES of CVB3, in directing translation initiation.     

In vitro translation of poliovirus RNA: utilization of internal initiation sites in reticulocyte lysate.

The translation of poliovirus RNA in rabbit reticulocyte lysate was examined. Translation of poliovirus RNA in this cell-free system resulted in an electrophoretic profile of poliovirus-specific proteins distinct from that observed in vivo or after translation in poliovirus-infected HeLa cell extract. A group of proteins derived from the P3 region of the polyprotein was identified by immunoprecipitation, time course, and N-formyl-[35S]methionine labeling studies to be the product of the initiation of protein synthesis at an internal site(s) located within the 3'-proximal RNA sequences. Utilization of this internal initiation site(s) on poliovirus RNA was abolished when reticulocyte lysate was supplemented with poliovirus-infected HeLa cell extract. Authentic P1-1a was also synthesized in reticulocyte lysate, indicating that correct 5'-proximal initiation of translation occurs in that system. We conclude that the deficiency of a component(s) of the reticulocyte lysate necessary for 5'-proximal initiation of poliovirus protein synthesis resulted in the ability of ribosomes to initiate translation on internal sequences. This aberrant initiation could be corrected by factors present in the HeLa cell extract. Apparently, under certain conditions, ribosomes are capable of recognizing internal sequences as authentic initiation sites.     

mRNAs containing the unstructured 5' leader sequence of alfalfa mosaic virus RNA 4 translate inefficiently in lysates from poliovirus-infected HeLa cells.

Poliovirus infection is accompanied by translational control that precludes translation of 5'-capped mRNAs and facilitates translation of the uncapped poliovirus RNA by an internal initiation mechanism. Previous reports have suggested that the capped alfalfa mosaic virus coat protein mRNA (AIMV CP RNA), which contains an unstructured 5' leader sequence, is unusual in being functionally active in extracts prepared from poliovirus-infected HeLa cells (PI-extracts). To identify the cis-acting nucleotide elements permitting selective AIMV CP expression, we tested capped mRNAs containing structured or unstructured 5' leader sequences in addition to an mRNA containing the poliovirus internal ribosome entry site (IRES). Translations were performed with PI-extracts and extracts prepared from mock-infected HeLa cells (MI-extracts). A number of control criteria demonstrated that the HeLa cells were infected by poliovirus and that the extracts were translationally active. The data strongly indicate that translation of RNAs lacking an internal ribosome entry site, including AIMV CP RNA, was severely compromised in PI-extracts, and we find no evidence that the unstructured AIMV CP RNA 5' leader sequence acts in cis to bypass the poliovirus translational control. Nevertheless, cotranslation assays in the MI-extracts demonstrate that mRNAs containing the unstructured AIMV CP RNA 5' untranslated region have a competitive advantage over those containing the rabbit alpha-globin 5' leader. Previous reports of AIMV CP RNA translation in PI-extracts likely describe inefficient expression that can be explained by residual cap-dependent initiation events, where AIMV CP RNA translation is competitive because of a diminished quantitative requirement for initiation factors.     

Direct evidence that polypyrimidine tract binding protein (PTB) is essential for internal initiation of translation of encephalomyocarditis virus RNA.

The requirement of PTB, polypyrimidine tract binding protein, for internal initiation of translation has been tested using an RNA affinity column to deplete rabbit reticulocyte lysates of PTB. The affinity column was prepared by coupling CNBr-activated Sepharose with the segment of the 5'-untranslated region of encephalomyocarditis virus (EMCV) RNA previously shown to bind PTB. Lysates passed through this column were devoid of PTB, and were incapable of internal initiation of translation dependent on the EMCV 5'-untranslated region, while retaining the capacity for translation dependent on ribosome scanning. Full activity for internal initiation was restored by the addition of recombinant PTB at the physiologically relevant concentration of about 5 micrograms/mL. When various PTB deletion mutants were tested, it was found that this activity required virtually the full-length protein. Thus, PTB is an essential protein for internal initiation promoted by the EMCV 5'-untranslated region. However, the PTB-depleted lysate retained the capacity for internal initiation promoted by the 5'-untranslated regions of another cardiovirus, Theiler's murine encephalomyelitis virus, and of the unrelated hepatitis C virus, and in neither case did addition of recombinant PTB stimulate internal initiation. Therefore, PTB is not a universal internal initiation factor that is indispensable in every case of internal ribosome entry.     

NMD resulting from encephalomyocarditis virus IRES-directed translation initiation seems to be restricted to CBP80/20-bound mRNA

Nonsense-mediated messenger RNA decay (NMD) generally degrades mRNAs that prematurely terminate translation as a means of quality control. NMD in mammalian cells targets newly spliced mRNA that is bound by the cap-binding protein heterodimer CBP80/20 and one or more post-splicing exon junction complexes during a pioneer round of translation. NMD targets mRNA that initiates translation using the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), therefore NMD might target not only CBP80/20-bound mRNA but also its remodelled product, eIF4E-bound mRNA. Here, we provide evidence that NMD triggered by translation initiation at the EMCV IRES, similar to NMD triggered by translation initiation at an mRNA cap, targets CBP80/20-bound mRNA but does not detectably target eIF4E-bound mRNA. We show that EMCV IRES-initiated translation undergoes a CBP80/20-associated pioneer round of translation that results in CBP80/20-dependent and Upf factor-dependent NMD when translation terminates prematurely.     

Differential cleavage of eIF4GI and eIF4GII in mammalian cells. Effects on translation

Two isoforms of the translation initiation factor eIF4G, eIF4GI and eIF4GII, have been described in eukaryotic cells. The exact function of each isoform during the initiation of protein synthesis is still under investigation. We have developed an efficient and reliable method of expressing poliovirus 2Apro, which differentially proteolyzes eIF4GI and eIF4GII in a time- and dose-dependent manner. This system is based on the electroporation of an in vitro transcribed mRNA that contains the encephalomyocarditis virus internal ribosome entry site followed by the sequence of poliovirus 2Apro. In contrast to HeLa cells, expression of this protease in BHK-21 cells induces delayed hydrolysis kinetics of eIF4GI with respect to eIF4GII. Moreover, under these conditions the polyadenylate binding protein is not cleaved. Interestingly, translation of de novo synthesized luciferase mRNA is highly dependent on eIF4GI integrity, whereas ongoing translation is inhibited at the same time as eIF4GII cleavage. Moreover, reinitiation of a preexisting mRNA translation after polysome run-off is dependent on the integrity of eIF4GII. Notably, de novo translation of heat shock protein 70 mRNA depends little on eIF4GI integrity but is more susceptible to eIF4GII hydrolysis. Finally, translation of an mRNA containing encephalomyocarditis virus internal ribosome entry site when the two isoforms of eIF4G are differentially hydrolyzed has been examined.     

The 5' untranslated region of the maize alcohol dehydrogenase gene contains an internal ribosome entry site

Adh1, the maize gene encoding alcohol dehydrogenase ADH1, mRNA is efficiently translated in O2-deprived roots of maize, whereas many normal cellular mRNAs are poorly translated. It has been shown that adh, the 5' untranslated region of adh1 mRNA, provides effective translation of mRNA under hypoxia and heat shock conditions in Nicotiana benthamiana plants. We found that adh contains the internal ribosome entry site (IRES) active both in vivo, in N. benthamiana cells, and in vitro, in rabbit reticulocyte lysate translation system. It is widely supposed that cap-independent internal initiation may maintain efficient translation of particular cellular mRNAs under a variety of stresses and other special conditions when cap-dependent protein synthesis is impaired. We evaluated the level of IRES activity of adh and found that its contribution to the overall translation of adh-containing mRNA in plant cells is less than 1% both under normal conditions and under heat shock. The low efficiency of this IRES is inconsistent with its possible role as a main factor ensuring efficient translation of adh1 mRNA under stress conditions.     

Attenuation stem-loop lesions in the 5' noncoding region of poliovirus RNA: neuronal cell-specific translation defects.

The nucleotide at position 480 in the 5' noncoding region of the viral RNA genome plays an important role in directing the attenuation phenotype of the Sabin vaccine strain of poliovirus type 1. In vitro translation studies have shown that the attenuated viral genomes of the Sabin strains direct levels of viral protein synthesis lower than those of their neurovirulent counterparts. We previously described the isolation of pseudorevertant polioviruses derived from transfections of HeLa cells with genome-length RNA harboring an eight-nucleotide lesion in a stem-loop structure (stem-loop V) that contains the attenuation determinant at position 480 (A. A. Haller and B. L. Semler, J. Virol. 66:5075-5086, 1992). This stem-loop structure is a major component of the poliovirus internal ribosome entry site required for initiation of viral protein synthesis. The eight-nucleotide lesion (X472) was lethal for virus growth and gave rise only to viruses which had partially reverted nucleotides within the original substituted sequences. In this study, we analyzed two of the poliovirus revertants (X472RI and X472R2) for cell-type-specific growth properties. The X472RI and X472R2 RNA templates directed protein synthesis to wild-type levels in in vitro translation reaction mixtures supplemented with crude cytoplasmic HeLa cell extracts. In contrast, the same X472 revertant RNAs displayed a decreased translation initiation efficiency when translated in a cell-free system supplemented with extracts from neuronal cells. This translation initiation defect of the X472R templates correlated with reduced yields of infectious virus particles in neuronal cells compared with those obtained from HeLa cells infected with the X472 poliovirus revertants. Our results underscore the important of RNA secondary structures within the poliovirus internal ribosome entry site in directing translation initiation and suggest that such structures interact with neuronal cell factors in a specific manner.     

Composition and arrangement of genes define the strength of IRES-driven translation in bicistronic mRNAs

In addition to the cap-dependent mechanism, eukaryotic initiation of translation can occur by a cap-independent mechanism which directs ribosomes to defined start codons enabled by internal ribosome entry site (IRES) elements. IRES elements from poliovirus and encephalomyocarditis virus are often used to construct bi- or oligocistronic expression vectors to co-express various genes from one mRNA. We found that while cap-dependent translation initiation from bicistronic mRNAs remains comparable to monocistronic expression, internal initiation mediated by these viral IRESs is often very inefficient. Expression of bicistronic expression vectors containing the hepatitis B virus core antigen (HBcAg) together with various cytokines in the second cistron of bicistronic mRNAs gave rise to very low levels of the tested cytokines. On the other hand, the HBcAg was well expressed when positioned in the second cistron. This suggests that the arrangement of cistrons in a bicistronic setting is crucial for IRES-dependent translation of the second cistron. A systematic examination of expression of reporter cistrons from bicistronic mRNAs with respect to position was carried out. Using the dual luciferase assay system we show that the composition of reading frames on a bicistronic mRNA and the order in which they are arranged define the strength of IRES-dependent translation. Although the cellular environment and the nature of the IRES element influence translation strength the dominant determinant is the nature and the arrangement of cistrons on the mRNA.     

The genome-linked protein VPg of the Norwalk virus binds eIF3, suggesting its role in translation initiation complex recruitment

The positive-strand RNA genomes of caliciviruses are not capped, but are instead covalently linked at their 5' ends to a viral protein called VPg. The lack of a cap structure typical of eukaryotic mRNA and absence of an internal ribosomal entry site suggest that VPg may function in translation initiation on calicivirus RNA. This hypothesis was tested by analyzing binding of Norwalk virus VPg to translation initiation factors. The eIF3d subunit of eIF3 was identified as a binding partner of VPg by yeast two-hybrid analysis. VPg bound to purified mammalian eIF3 and to eIF3 in mammalian cell lysates. To test the effects of the VPg- eIF3 interaction on translation, VPg was added to cell-free translation reactions programmed with either capped reporter RNA, an RNA containing an EMCV internal ribosomal entry site (IRES) or an RNA with a cricket paralysis virus IRES. VPg inhibited translation of all reporter RNAs in a dose-dependent manner. Together, the data suggest that VPg may play a role in initiating translation on calicivirus RNA through unique protein-protein interactions with the translation machinery.     

Unique features of internal initiation of hepatitis C virus RNA translation.

The question of whether hepatitis C virus (HCV) RNA is translated by a mechanism of internal ribosome entry has been examined by testing whether insertion of HCV sequences between the two cistrons of a dicistronic mRNA promotes translation of the downstream cistron in rabbit reticulocyte lysates. Deletion analysis showed that efficient internal initiation required a segment of the HCV genome extending from about nucleotides 40-370 and that deletions from the 3'-end of this element were highly deleterious. As the authentic initiation codon for HCV polyprotein synthesis is at nucleotide 342, this demonstrates that, besides 5'-UTR sequences, a short length of HCV coding sequences is required for internal initiation. This finding was confirmed in transfection assays of BT7-H cells and was shown to be independent of the nature of the downstream reporter cistron. The strong requirement for coding sequences is in sharp contrast to internal initiation of picornavirus RNA translation. As a probable correlate with this, it was also found that the efficiency of internal initiation was only marginally compromised when the authentic initiation codon was mutated to a non-AUG codon, again in sharp contrast with the picornaviruses. The finding that coding sequences are required for internal initiation has important implications for the design of experiments to test for internal initiation of translation of cellular mRNAs.     

Construction of regulatable picornavirus IRESes as a test of current models of the mechanism of internal translation initiation

Picornavirus internal ribosome entry sites (IRESs) are approximately 450 nt. RNA elements that direct internal initiation of translation, such that when placed between the two cistrons of a dicistronic construct, they drive independent translation of the downstream cistron. Consequently they have been widely used for coordinated expression of two or more proteins. All picornavirus IRESs have an AUG triplet at the very 3' end, which is thought to be the actual site of internal ribosome entry. However with some IRESs, such as foot-and-mouth disease virus, and especially poliovirus, the majority of ribosomes do not initiate translation at this putative entry site AUG, but at the next AUG further downstream, which is thought to be accessed by a process of linear ribosome scanning from the entry site. If this is so, then it should be possible to regulate IRES-dependent translation by inserting an iron responsive element (IRE) between the putative entry site AUG and the main functional initiation site. This should make IRES-dependent translation sensitive to the concentration of iron regulatory protein (IRP), the protein that specifically binds to the IRE. This has been attempted with both the foot-and-mouth disease virus and poliovirus IRESs, and was successful in so far as an inhibition specifically of IRES-dependent translation was observed that was strictly dependent on both the presence of IRP and of a functional IRE motif inserted in the sense orientation. However, the range over which expression could be varied was rather limited (three- to fourfold maximum), because some IRES-dependent translation remained completely refractory to inhibition by even very high IRP concentrations. In contrast, with a cap-proximal IRE in the 5' untranslated region of an mRNA translated by the scanning mechanism, addition of sufficient IRP results in complete inhibition. These results support the model of IRES-promoted ribosome entry at an upstream site followed by strictly linear scanning to the main functional initiation site for the majority of internal initiation events, but imply that some ribosomes must access the functional initiation site by another route, possibly a nonlinear shunting-like mechanism.     

Hepatitis C virus internal ribosome entry site-dependent translation in Saccharomyces cerevisiae is independent of polypyrimidine tract-binding protein, poly(rC)-binding protein 2, and La protein

Translation initiation of some viral and cellular mRNAs occurs by ribosome binding to an internal ribosome entry site (IRES). Internal initiation mediated by the hepatitis C virus (HCV) IRES in Saccharomyces cerevisiae was shown by translation of the second open reading frame in a bicistronic mRNA. Introduction of a single base change in the HCV IRES, known to abrogate internal initiation in mammalian cells, abolished translation of the second open reading frame. Internal initiation mediated by the HCV IRES was independent of the nonsense-mediated decay pathway and the cap binding protein eIF4E, indicating that translation is not a result of mRNA degradation or 5'-end-dependent initiation. Human La protein binds the HCV IRES and is required for efficient internal initiation. Disruption of the S. cerevisiae genes that encode La protein orthologs and synthesis of wild-type human La protein in yeast had no effect on HCV IRES-dependent translation. Polypyrimidine tract-binding protein (Ptb) and poly-(rC)-binding protein 2 (Pcbp2), which may be required for HCV IRES-dependent initiation in mammalian cells, are not encoded within the S. cerevisiae genome. HCV IRES-dependent translation in S. cerevisiae was independent of human Pcbp2 protein and stimulated by the presence of human Ptb protein. These findings demonstrate that the genome of S. cerevisiae encodes all proteins necessary for internal initiation of translation mediated by the HCV IRES.