Regulation of internal ribosomal entry site-mediated translation by phosphorylation of the translation initiation factor eIF2alpha

Initiation of translation from most cellular mRNAs occurs via scanning; the 40 S ribosomal subunit binds to the m(7)G-cap and then moves along the mRNA until an initiation codon is encountered. Some cellular mRNAs contain internal ribosome entry sequences (IRESs) within their 5'-untranslated regions, which allow initiation independently of the 5'-cap. This study investigated the ability of cellular stress to regulate the activity of IRESs in cellular mRNAs. Three stresses were studied that cause the phosphorylation of the translation initiation factor, eIF2alpha, by activating specific kinases: (i) amino acid starvation, which activates GCN2; (ii) endoplasmic reticulum (ER) stress, which activates PKR-like ER kinase, PERK kinase; and (iii) double-stranded RNA, which activates double-stranded RNA-dependent protein kinase (PKR) by mimicking viral infection. Amino acid starvation and ER stress caused transient phosphorylation of eIF2alpha during the first hour of treatment, whereas double-stranded RNA caused a sustained phosphorylation of eIF2alpha after 2 h. The effects of these treatments on IRES-mediated initiation were investigated using bicistronic mRNA expression vectors. No effect was seen for the IRESs from the mRNAs for the chaperone BiP and the protein kinase Pim-1. In contrast, translation mediated by the IRESs from the cationic amino acid transporter, cat-1, and of the cricket paralysis virus intergenic region, were stimulated 3- to 10-fold by all three treatments. eIF2alpha phosphorylation was required for the response because inactivation of phosphorylation prevented the stimulation. It is concluded that cellular stress can stimulate translation from some cellular IRESs via a mechanism that requires the phosphorylation of eIF2alpha. Moreover, there are distinct regulatory patterns for different cellular mRNAs that contain IRESs within their 5'-untranslated regions.     

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.     

Rational construction of eukaryotic OFF-riboswitches that downregulate internal ribosome entry site-mediated translation in response to their ligands

A strategy for rationally constructing a novel type of eukaryotic OFF-riboswitch, which ligand-dependently turns off translation mediated by an internal ribosome entry site (IRES), has been established. The theophylline-dependent IRES-based OFF-riboswitch obtained through the proposed strategy functioned well in wheat germ extract, independently from the downstream gene, indicating that it can regulate any gene. Despite the fact that it has one theophylline aptamer, its switching efficiency was as high as that of a previously reported theophylline-dependent OFF-riboswitch that was constructed by inserting three continuous theophylline aptamers into a 5' untranslated region in mRNA to downregulate the normal 5'-terminus-mediated translation. In addition, because the riboswitch part that was optimized in the theophylline-dependent IRES-based OFF-riboswitch, except for the aptamer domain, can be used as-is for other aptamer-ligand pairs, an arbitrary ligand-dependent IRES-based OFF-riboswitch is easy to construct with the corresponding well-minimized aptamer.     

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.     

Dual targeting of Arabidopsis holocarboxylase synthetase1: a small upstream open reading frame regulates translation initiation and protein targeting

Protein biotinylation is an original and very specific posttranslational modification, compartmented in plants, between mitochondria, plastids, and the cytosol. This reaction modifies and activates few carboxylases committed in key metabolisms and is catalyzed by holocarboxylase synthetase (HCS). The molecular bases of this complex compartmentalization and the relative function of each of the HCS genes, HCS1 and HCS2, identified in Arabidopsis (Arabidopsis thaliana) are mainly unknown. Here, we showed by reverse genetics that the HCS1 gene is essential for plant viability, whereas disruption of the HCS2 gene in Arabidopsis does not lead to any obvious phenotype when plants are grown under standard conditions. These findings strongly suggest that HCS1 is the only protein responsible for HCS activity in Arabidopsis cells, including the cytosolic, mitochondrial, and plastidial compartments. A closer study of HCS1 gene expression enabled us to propose an original mechanism to account for this multiplicity of localizations. Located in the HCS1 messenger RNA 5'-untranslated region, an upstream open reading frame regulates the translation initiation of HCS1 and the subsequent targeting of HCS1 protein. Moreover, an exquisitely precise alternative splicing of HCS1 messenger RNA can regulate the presence and absence of this upstream open reading frame. The existence of these complex and interdependent mechanisms creates a rich molecular platform where different parameters and factors could control HCS targeting and hence biotin metabolism.     

Demonstration of functional requirement of polypyrimidine tract-binding protein by SELEX RNA during hepatitis C virus internal ribosome entry site-mediated translation initiation

Polypyrimidine tract-binding protein (PTB) has been previously shown to physically interact with the hepatitis C virus (HCV) RNA genome at its 5'- and 3'-noncoding regions. Using high affinity SELEX RNA molecules, we present evidence for the functional requirement of PTB during HCV internal ribosome entry site (IRES)-controlled translation initiation. This study was carried out in rabbit reticulocyte translation lysates in which the HCV IRES-driven reporter RNA was introduced along with the PTB-specific SELEX RNA molecules. The SELEX RNAs specifically inhibited the HCV IRES function in the context of mono- and dicistronic mRNAs. The cap-dependent translation of a reporter (chloramphenicol acetyltransferase) RNA or naturally capped brome mosaic virus RNA, however, was not affected by the presence of SELEX during in vitro translation assays. The SELEX-mediated inhibition of the HCV IRES is shown to be relieved by the addition of recombinant human PTB in an add-back experiment. The in vivo requirement of PTB was further confirmed by cotransfection of Huh7 cells with reporter RNA and PTB-specific SELEX RNA. The HCV IRES activity was inhibited by the SELEX RNA in these cells, but not by an unrelated control RNA. Together, these results demonstrate the functional requirement of cellular PTB in HCV translation and further support the feasible use of SELEX RNA strategy in demonstrating the functional relevance of cellular protein(s) in complex biological processes.     

Picornavirus internal ribosome entry site elements can stimulate translation of upstream genes

Certain viral and cellular mRNAs initiate translation cap-independently at internal ribosome entry site (IRES) elements. Picornavirus IRES elements are widely used in dicistronic or multicistronic vectors in gene therapy, virus replicon systems, and analysis of IRES function. In such vectors, expression of the upstream gene often serves as internal control to standardize the readings of IRES-driven downstream reporter activity. Picornaviral IRES elements translate optimally at up to 120 mM K(+) concentration, whereas genes used as upstream reporters usually have lower salt optima when present in monocistronic mRNAs. However, here we show that such reporter genes are efficiently translated at higher K(+) concentrations when placed upstream of a functional picornavirus IRES. This translation enhancement occurs in cis, is independent of the nature of the first reporter and of second reporter translation, and is conferred by the IRESs of picornaviruses but not of hepatitis C virus. A defective picornavirus IRES with a deletion killing IRES activity but leaving the binding site for initiation factor eIF4G intact retains translation enhancement activity. Translation enhancement on a capped mRNA is disabled by m(7)GDP. In addition, the C-terminal fragment of eIF4G can confer translation enhancement also on uncapped mRNA. We conclude that whenever eIF4F has been captured to a dicistronic mRNA by binding to a picornavirus IRES via its eIF4G moiety, it can be provided in cis to the 5'-end of the RNA and there stimulate translation initiation, either by binding to the cap nucleotide using its eIF4E moiety or by binding to the RNA cap-independently using its eIF4G moiety.     

Hepatitis C virus internal ribosome entry site-mediated translation is stimulated by cis-acting RNA elements and trans-acting viral factors

Translation initiation of hepatitis C virus (HCV) occurs through an internal ribosome entry site (IRES) located at its 5'-end. As a positive-stranded RNA virus, HCV uses its genome as a common template for translation and replication, but the coordination between these two processes remains poorly characterized. Moreover, although genetic evidence of RNA-protein interactions for viral replication is accumulating because of subgenomic replicons and a recent culture system for HCV, such interactions are still contentious in the regulation of translation. To gain insight into such mechanisms, we addressed the involvement of cis and trans viral factors in HCV IRES activity by using a cell-based RNA reporter system. We found that the HCV 3' noncoding region (NCR) strongly stimulates IRES efficiency in cis, depending on the genotype and the cell line. Moreover, we confirmed the role of the core protein in viral gene expression as previously reported in vitro. Surprisingly, we observed a similar effect, i.e. a twofold increase under low amounts of NS5B RNA polymerase, followed by a decrease at higher concentrations. However, no contribution of NS5A to HCV IRES-mediated translation was noted and no cooperative effect could be detected between 3' NCR and viral proteins or between proteins. Collectively, these results suggest that HCV RNA translation is regulated, and that the switch from translation to replication might involve a sequential requirement for both cis and trans viral factors, because of their apparent lack of synergy, probably with the aid of host factors.     

Tumor suppressor PDCD4 represses internal ribosome entry site-mediated translation of antiapoptotic proteins and is regulated by S6 kinase 2

Apoptosis can be regulated by extracellular signals that are communicated by peptides such as fibroblast growth factor 2 (FGF-2) that have important roles in tumor cell proliferation. The prosurvival effects of FGF-2 are transduced by the activation of the ribosomal protein S6 kinase 2 (S6K2), which increases the expression of the antiapoptotic proteins X chromosome-linked Inhibitor of Apoptosis (XIAP) and Bcl-x(L). We now show that the FGF-2-S6K2 prosurvival signaling is mediated by the tumor suppressor programmed cell death 4 (PDCD4). We demonstrate that PDCD4 specifically binds to the internal ribosome entry site (IRES) elements of both the XIAP and Bcl-x(L) messenger RNAs and represses their translation by inhibiting the formation of the 48S translation initiation complex. Phosphorylation of PDCD4 by activated S6K2 leads to the degradation of PDCD4 and thus the subsequent derepression of XIAP and Bcl-x(L) translation. Our results identify PDCD4 as a specific repressor of the IRES-dependent translation of cellular mRNAs (such as XIAP and Bcl-x(L)) that mediate FGF-2-S6K2 prosurvival signaling and provide further insight into the role of PDCD4 in tumor suppression.     

Age-dependent poliovirus replication in the mouse central nervous system is determined by internal ribosome entry site-mediated translation

Mouse cells are not permissive for the replication of human rhinovirus type 2 (HRV2). To determine the role of the HRV2 internal ribosome entry site (IRES) in determining species specificity, a recombinant poliovirus (P1/HRV2) was constructed by substituting the poliovirus IRES with the IRES from HRV2. This recombinant virus replicated in all human and murine cell lines examined, demonstrating that the HRV2 IRES does not limit viral replication in transformed murine cells. P1/HRV2 replicated in the brain and spinal cord in neonatal but not adult mice transgenic for the poliovirus receptor, CD155. Passage of P1/HRV2 in mice led to selection of a virus that caused paralysis in neonatal mice. To determine the relationship between HRV2 IRES-mediated translation and replication of P1/HRV2 in mice, recombinant human adenoviruses were used to express bicistronic mRNAs in murine organs. The results demonstrate that the HRV2 IRES mediates translation in organs of neonatal but not adult mice. These findings show that HRV2 IRES-mediated translation is a determinant of virus replication in the murine brain and spinal cord and suggest that the IRES determines the species specificity of HRV2 infection.     

An internal ribosome entry site mediates translation of lymphoid enhancer factor-1

The lymphoid enhancer factor-1 LEF1 locus produces multiple mRNAs via alternative promoters. Full-length LEF-1 protein is produced via translation of an mRNA with a 1.2-kb, GC-rich 5'-untranslated region (UTR), whereas a truncated LEF-1 isoform is produced by an mRNA with a short, 60-nucleotide (nt) 5'-UTR. Full-length LEF-1 promotes cell growth via its interaction with the WNT signaling mediator beta-catenin. Truncated LEF-1 lacks the beta-catenin binding domain and opposes WNT signaling as a competitive inhibitor for WNT response elements. In this study we tested the hypothesis that the long, GC-rich 5'-UTR within the full-length LEF1 mRNA contains an internal ribosome entry site (IRES). Using a dicistronic vector in transient DNA transfections, we show that the LEF1 5'-UTR mediates cap-independent translation. Additional experiments involving a promoter-less dicistronic vector, Northern blot analysis, and transient transfections of dicistronic mRNAs into cultured mammalian cells compromised for cap-dependent translation demonstrate that the 5'-UTR of full-length LEF1 mRNA contains a bona fide IRES. Deletion analysis of the 5'-UTR shows that maximal IRES activity requires the majority of the 5'-UTR, consistent with the notion that cellular IRESs require multiple modules for efficient activity. This study demonstrates that full-length LEF1 mRNA has evolved to utilize a cap-independent mechanism for translation of full-length LEF-1, whereas the truncated isoform is produced via the canonical cap-dependent ribosome scanning mechanism.     

La autoantigen is required for the internal ribosome entry site-mediated translation of Coxsackievirus B3 RNA

Translation initiation in Coxsackievirus B3 (CVB3) occurs via ribosome binding to an internal ribosome entry site (IRES) located in the 5′-untranslated region (UTR) of the viral RNA. This unique mechanism of translation initiation requires various trans-acting factors from the host. We show that human La autoantigen (La) binds to the CVB3 5′-UTR and also demonstrate the dose-dependent effect of exogenously added La protein in stimulating CVB3 IRES-mediated translation. The requirement of La for CVB3 IRES mediated translation has been further demonstrated by inhibition of translation as a result of sequestering La and its restoration by exogenous addition of recombinant La protein. The abundance of La protein in various mouse tissue extracts has been probed using anti-La antibody. Pancreatic tissue, a target organ for CVB3 infection, was found to have a large abundance of La protein which was demonstrated to interact with the CVB3 5′-UTR. Furthermore, exogenous addition of pancreas extract to in vitro translation reactions resulted in a dose dependent stimulation of CVB3 IRES-mediated translation. These observations indicate the role of La in CVB3 IRES-mediated translation, and suggest its possible involvement in the efficient translation of the viral RNA in the pancreas.