Internal Ribosome Entry Site-mediated Translation Research Articles

Regulation of Internal Ribosome Entry Site-mediated Translation by Eukaryotic Initiation Factor-2α Phosphorylation and Translation of a Small Upstream Open Reading Frame

Adaptation to amino acid deficiency is critical for cell survival. In yeast, this adaptation involves phosphorylation of the translation eukaryotic initiation factor (eIF) 2alpha by the kinase GCN2. This leads to the increased translation of the transcription factor GCN4, which in turn increases transcription of amino acid biosynthetic genes, at a time when expression of most genes decreases. Here it is shown that translation of the arginine/lysine transporter cat-1 mRNA increases during amino acid starvation of mammalian cells. This increase requires both GCN2 phosphorylation of eIF2alpha and the translation of a 48-amino acid upstream open reading frame (uORF) present within the 5'-leader of the transporter mRNA. When this 5'-leader was placed in a bicistronic mRNA expression vector, it functioned as an internal ribosomal entry sequence and its regulated activity was dependent on uORF translation. Amino acid starvation also induced translation of monocistronic mRNAs containing the cat-1 5'-leader, in a manner dependent on eIF2alpha phosphorylation and translation of the 48-amino acid uORF. This is the first example of mammalian regulation of internal ribosomal entry sequence-mediated translation by eIF2alpha phosphorylation during amino acid starvation, suggesting that the mechanism of induced Cat-1 protein synthesis is part of the adaptive response of cells to amino acid limitation.

Involvement of proteasome alpha-subunit PSMA7 in hepatitis C virus internal ribosome entry site-mediated translation.

Ribozymes are small catalytic RNA molecules that can be engineered to enzymatically cleave RNA transcripts in a sequence-specific fashion and thereby inhibit expression and function of the corresponding gene product. With their simple structures and site-specific cleavage activity, they have been exploited as potential therapeutic agents in a variety of human disorders, including hepatitis C virus (HCV) infection. We have designed a hairpin ribozyme (Rz3'X) targeting the HCV minus-strand replication intermediate at position 40 within the 3'X tail. Surprisingly, Rz3'X was found to induce ganciclovir (GCV)-resistant colonies in a bicistronic cellular reporter system with HCV internal ribosome entry site (IRES)-dependent translation of herpes simplex virus thymidine kinase (TK). Rz3'X-transduced GCV-resistant HeLa reporter cells showed substantially reduced IRES-mediated HCV core protein translation compared with control vector-transduced cells. Since these reporter systems do not contain the HCV 3'X tail sequences, the results indicate that Rz3'X probably exerted an inhibitory effect on HCV IRES activity fortuitously through another gene target. A novel technique of ribozyme cleavage-based target gene identification (cleavage-specific amplification of cDNA ends) (M. Krüger, C. Beger, P. J. Welch, J. R. Barber, and F. Wong-Staal, Nucleic Acids Res. 29:e94, 2001) revealed that human 20S proteasome alpha-subunit PSMA7 mRNA was a target RNA recognized and cleaved by Rz3'X. We then showed that additional ribozymes directed against PSMA7 RNA inhibited HCV IRES activity in two assay systems: GCV resistance in the HeLa IRES TK reporter cell system and a transient transfection assay performed with a bicistronic Renilla-HCV IRES-firefly luciferase reporter in Huh7 cells. In contrast, ribozymes were inactive against IRES of encephalomyocarditis virus and human rhinovirus. Additionally, proteasome inhibitor MG132 exerted a dose-dependent inhibitory effect on HCV IRES-mediated translation but not on cap-dependent translation. These data suggest a principal role for PSMA7 in regulating HCV IRES activity, a function essential for HCV replication.

Internal Ribosome Entry Site-mediated Translation of a Mammalian mRNA Is Regulated by Amino Acid Availability

The cationic amino acid transporter, Cat-1, facilitates the uptake of the essential amino acids arginine and lysine. Amino acid starvation causes accumulation and increased translation of cat-1 mRNA, resulting in a 58-fold increase in protein levels and increased arginine uptake. A bicistronic mRNA expression system was used to demonstrate the presence of an internal ribosomal entry sequence (IRES) within the 5'-untranslated region of the cat-1 mRNA. This study shows that IRES-mediated translation of the cat-1 mRNA is regulated by amino acid availability. This IRES causes an increase in translation under conditions of amino acid starvation. In contrast, cap-dependent protein synthesis is inhibited during amino acid starvation, which is well correlated with decreased phosphorylation of the cap-binding protein, eIF4E. These findings reveal a new aspect of mammalian gene expression and regulation that provides a cellular stress response; when the nutrient supply is limited, the activation of IRES-mediated translation of mammalian mRNAs results in the synthesis of proteins essential for cell survival.

Recognition of nascent RNA by the human La antigen: conserved and divergent features of structure and function.

La is a conserved RNA-binding phosphoprotein that interacts with a large variety of ligands. The most ubiquitous function of La is association with newly synthesized RNA polymerase (Pol) III transcripts via their common UUU-OH 39 termini and stabilization of these against exonucleolytic digestion. Accumulating evidence also indicates an activity for La in internal ribosome entry site-mediated translation in mammalian cells and in the metabolism of a subset of 39-processed snRNA intermediates that end in uridylates but are synthesized by Pol II. The most highly conserved region of La resides in the N-terminal domain (NTD), and this appears to mediate highaffinity UUU-OH recognition. As critically reviewed here by comparison to a consensus core RNA recognition motif (RRM) structure, the NTD can be modeled into a pair of tandem RRMs. In addition to the conserved NTD, human La (hLa) protein contains a C-terminal domain (CTD) that harbors a third RRM and a potential Walker A motif that appears to recognize the 59-ppp ends of nascent RNAs. The resulting bipartite mode of RNA binding can account for previously unexplained observations and may underlie a unifying principle of La function. While a role for hLa in transcription remains controversial, its presence in a Pol III holoenzyme suggests a role reminiscent of the CTD of Pol II, as an integrator of transcriptional and posttranscriptional activities that include 59- and 39-RNA metabolism. Evidence that the 59-end-RNA recognition activity of hLa can be modulated by phosphorylation provides mechanistic insight into the signal transduction

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.

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.

Methionine-independent initiation of translation in the capsid protein of an insect RNA virus.

Protein synthesis is believed to be initiated with the amino acid methionine because the AUG translation initiation codon of mRNAs is recognized by the anticodon of initiator methionine transfer RNA. A group of positive-stranded RNA viruses of insects, however, lacks an AUG translation initiation codon for their capsid protein gene, which is located at the downstream part of the genome. The capsid protein of one of these viruses, Plautia stali intestine virus, is synthesized by internal ribosome entry site-mediated translation. Here we report that methionine is not the initiating amino acid in the translation of the capsid protein in this virus. Its translation is initiated with glutamine encoded by a CAA codon that is the first codon of the capsid-coding region. The nucleotide sequence immediately upstream of the capsid-coding region interacts with a loop segment in the stem-loop structure located 15-43 nt upstream of the 5' end of the capsid-coding region. The pseudoknot structure formed by this base pair interaction is essential for translation of the capsid protein. This mechanism for translation initiation differs from the conventional one in that the initiation step controlled by the initiator methionine transfer RNA is not necessary.

A Novel Form of DAP5 Protein Accumulates in Apoptotic Cells as a Result of Caspase Cleavage and Internal Ribosome Entry Site-Mediated Translation

Death-associated protein 5 (DAP5) (also named p97 and NAT1) is a member of the translation initiation factor 4G (eIF4G) family that lacks the eIF4E binding site. It was previously implicated in apoptosis, based on the finding that a dominant negative fragment of the protein protected against cell death. Here we address its function and two distinct levels of regulation during apoptosis that affect the protein both at translational and posttranslational levels. DAP5 protein was found to be cleaved at a single caspase cleavage site at position 790, in response to activated Fas or p53, yielding a C-terminal truncated protein of 86 kDa that is capable of generating complexes with eIF4A and eIF3. Interestingly, while the overall translation rate in apoptotic cells was reduced by 60 to 70%, in accordance with the simultaneous degradation of the two major mediators of cap-dependent translation, eIF4GI and eIF4GII, the translation rate of DAP5 protein was selectively maintained. An internal ribosome entry site (IRES) element capable of directing the translation of a reporter gene when subcloned into a bicistronic vector was identified in the 5' untranslated region of DAP5 mRNA. While cap-dependent translation from this transfected vector was reduced during Fas-induced apoptosis, the translation via the DAP5 IRES was selectively maintained. Addition of recombinant DAP5/p97 or DAP5/p86 to cell-free systems enhanced preferentially the translation through the DAP5 IRES, whereas neutralization of the endogenous DAP5 in reticulocyte lysates by adding a dominant negative DAP5 fragment interfered with this translation. The DAP5/p86 apoptotic form was more potent than DAP5/p97 in these functional assays. Altogether, the data suggest that DAP5 is a caspase-activated translation factor which mediates cap-independent translation at least from its own IRES, thus generating a positive feedback loop responsible for the continuous translation of DAP5 during apoptosis.

Internal ribosome entry site-mediated translation in hepatitis C virus replication.

The initiation of protein translation on eukaryotic messenger RNAs predominantly follows the first AUG rule, as described by KOZAK (1989b). This states that the ribosome begins scanning an RNA molecule from its extreme 5’ end until it encounters an AUG codon, at which point translation is initiated. In eukaryotes, mRNA molecules usually carry an m7GpppG cap structure at their 5’ terminus. This 5’ cap-structure strongly enhances translation, as it facilitates binding of translation initiation factors and the 40S ribosome subunit to the mRNA (reviewed by PAIN 1996; SACHS et al. 1997). The scanning process, or 3’ movement of the 40S ribosome subunit along the RNA in search of an AUG codon, is inhibited by very stable RNA structures. Also, AUG codons positioned between the 5’ terminus and the AUG codon initiating a specific coding region reduce the efficiency of translation.

Amantadine and rimantadine have no direct inhibitory effects against hepatitis C viral protease, helicase, ATPase, polymerase, and internal ribosomal entry site-mediated translation.

Amantadine, a drug known to inhibit influenza A viral matrix (M2) protein function, was reported to be an effective treatment in some patients with chronic hepatitis C virus (HCV) infection. Sequence comparison shows no homology between M2 and any of the HCV proteins. The effects of amantadine and a related analogue, rimantadine, on viral protease, helicase, ATPase, RNA-dependent RNA polymerase, and HCV internal ribosomal entry site (IRES) translation were tested by established in vitro biochemical assays. No inhibition (>15%) of HCV protease, helicase, ATPase, and polymerase was observed with concentrations up to 400 microgram/mL. IRES-specific inhibition was not observed at clinically relevant concentrations, but both cap and IRES reporter genes were suppressed at higher levels, suggesting nonspecific translation inhibition. In conclusion, amantadine and rimantadine have no direct and specific inhibitory effects against HCV protease, helicase, ATPase, polymerase, and IRES in vitro.

A coding RNA sequence acts as a replication signal in cardioviruses.

Theiler's virus and Mengo virus are representatives of the Cardiovirus genus within the picornavirus family. Their genome is an 8-kilobase long positive strand RNA molecule. This RNA molecule plays three roles in infected cells: It serves as a messenger RNA, acts as a template for genome replication, and is encapsidated to form progeny virions. We observed that a cis-acting signal required for replication of Theiler's virus was contained within a 130-nt stretch of the region encoding the capsid protein VP2. This RNA sequence does not influence internal ribosome entry site-mediated translation initiation and thus likely acts directly as a signal for the replication complex. We found a similar signal in the VP2-coding sequence of Mengo virus, and both signals could be functionally exchanged. Within the replication element, a 9-nt sequence that is highly conserved among cardioviruses was shown to be essential for replication. This conserved sequence was contained in mostly unpaired regions of the RNA secondary structure predicted for the replication elements of the various cardioviruses. Interestingly, a similar replication element has been reported to occur in the distantly related human rhinovirus type 14, suggesting that such elements could be conserved throughout the picornavirus family. However, the different location of the replication elements in rhinovirus and cardioviruses, and the fact that they were not functionally exchangeable, is raising intriguing questions about the evolution of such signals in picornaviruses.

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.

The La antigen binds 5' noncoding region of the hepatitis C virus RNA in the context of the initiator AUG codon and stimulates internal ribosome entry site-mediated translation.

Translation initiation of the hepatitis C virus (HCV) RNA genome occurs through an internal ribosome entry site in a cap-independent manner. Here, we have examined the interaction between La antigen and the HCV 5' noncoding region (5'NCR). In this analysis, competitor RNAs derived from HCV 5'NCR carrying deletions and a point mutation were used to identify the site(s) of La antigen binding during UV cross-linking assay. These studies suggest that La antigen recognizes the intact HCV 5'NCR structure. Further, these interactions occurred in the context of the initiator AUG. The latter view is supported by an analysis in which mutants of the HCV 5'NCR RNA with deletion or substitution in the initiator AUG codon failed to compete for La antigen binding to the wild-type 5'NCR. The evidence for the interaction between liver cell-derived La antigen and the HCV 5'NCR is provided by immunoprecipitation of a UV cross-linked species from the S100 fraction of Huh7 cell lysates. The functional relevance of this interaction was demonstrated by the stimulation of the HCV internal ribosome entry site-mediated translation in the presence of La protein. These results suggest an important functional role of La protein in the regulation of internal initiation of translation of the HCV RNA genome.