Translation of hepatitis C virus RNA

The 341-nucleotide 5' non-translated region is the most conserved part of the hepatitis C virus (HCV) genome. It contains a highly structured internal ribosomal entry site (IRES) that mediates cap-independent initiation of translation of the viral polyprotein by a mechanism that is unprecedented in eukaryotes. The first step in translation initiation is assembly of eukaryotic initiation factor (eIF) 3, eIF2, GTP, initiator tRNA and a 40S ribosomal subunit into a 43S preinitiation complex. The HCV IRES recruits this complex and directs its precise attachment at the initiation codon to form a 48S complex in a process that does not involve eIFs 4A, 4B or 4F. The IRES contains sites that bind independently with the eIF3 and 40S subunit components of 43S complexes, and structural determinants that ensure the correct spatial orientation of these binding sites so that the 48S complex assembles precisely at the initiation codon.     

Coordinated assembly of human translation initiation complexes by the hepatitis C virus internal ribosome entry site RNA

Protein synthesis in all cells begins with recruitment of the small ribosomal subunit to the initiation codon in a messenger RNA. In some eukaryotic viruses, RNA upstream of the coding region forms an internal ribosome entry site (IRES) that directly binds to the 40S ribosomal subunit and enables translation initiation in the absence of many canonical translation initiation factors. The hepatitis C virus (HCV) IRES RNA requires just two initiation factors, eukaryotic initiation factor (eIF) 2 and eIF3, to form preinitiation 48S ribosomal complexes that subsequently assemble into translation-competent ribosomes. Using an RNA-based affinity purification approach, we show here that HCV IRES RNA facilitates eIF2 function through its interactions with eIF3 and the 40S ribosomal subunit. Although the wild-type IRES assembles normally into 48S and 80S ribosomal complexes in human cell extract, mutant IRES RNAs become trapped at the 48S assembly stage. Trapped 48S complexes formed by IRES mutants with reduced eIF3 binding affinity nonetheless contain eIF3, consistent with inherent eIF3-40S subunit affinity. Intriguingly, however, one of these IRES mutants prevents stable association of both eIF3 and eIF2, preventing initiator tRNA deposition and explaining the block in 80S assembly. In contrast, an IRES mutant unable to induce a conformational change in the 40S subunit, as observed previously by single-particle cryoelectron microscopy, blocks 80S formation at a later stage in assembly. These data suggest that the IRES RNA coordinates interactions of eIF3 and eIF2 on the ribosome required to position the initiator tRNA on the mRNA in the ribosomal peptidyl-tRNA site (P site).     

Molecular mechanisms of translation initiation in eukaryotes

Translation initiation is a complex process in which initiator tRNA, 40S, and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of mRNA. The cap-binding complex eIF4F and the factors eIF4A and eIF4B are required for binding of 43S complexes (comprising a 40S subunit, eIF2/GTP/Met-tRNAi and eIF3) to the 5' end of capped mRNA but are not sufficient to promote ribosomal scanning to the initiation codon. eIF1A enhances the ability of eIF1 to dissociate aberrantly assembled complexes from mRNA, and these factors synergistically mediate 48S complex assembly at the initiation codon. Joining of 48S complexes to 60S subunits to form 80S ribosomes requires eIF5B, which has an essential ribosome-dependent GTPase activity and hydrolysis of eIF2-bound GTP induced by eIF5. Initiation on a few mRNAs is cap-independent and occurs instead by internal ribosomal entry. Encephalomyocarditis virus (EMCV) and hepatitis C virus epitomize distinct mechanisms of internal ribosomal entry site (IRES)-mediated initiation. The eIF4A and eIF4G subunits of eIF4F bind immediately upstream of the EMCV initiation codon and promote binding of 43S complexes. EMCV initiation does not involve scanning and does not require eIF1, eIF1A, and the eIF4E subunit of eIF4F. Initiation on some EMCV-like IRESs requires additional noncanonical initiation factors, which alter IRES conformation and promote binding of eIF4A/4G. Initiation on the hepatitis C virus IRES is even simpler: 43S complexes containing only eIF2 and eIF3 bind directly to the initiation codon as a result of specific interaction of the IRES and the 40S subunit.     

Functional reconstitution of human eukaryotic translation initiation factor 3 (eIF3)

Protein fate in higher eukaryotes is controlled by three complexes that share conserved architectural elements: the proteasome, COP9 signalosome, and eukaryotic translation initiation factor 3 (eIF3). Here we reconstitute the 13-subunit human eIF3 in Escherichia coli, revealing its structural core to be the eight subunits with conserved orthologues in the proteasome lid complex and COP9 signalosome. This structural core in eIF3 binds to the small (40S) ribosomal subunit, to translation initiation factors involved in mRNA cap-dependent initiation, and to the hepatitis C viral (HCV) internal ribosome entry site (IRES) RNA. Addition of the remaining eIF3 subunits enables reconstituted eIF3 to assemble intact initiation complexes with the HCV IRES. Negative-stain EM reconstructions of reconstituted eIF3 further reveal how the approximately 400 kDa molecular mass structural core organizes the highly flexible 800 kDa molecular mass eIF3 complex, and mediates translation initiation.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site

Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.Protein synthesis is a central process in health and disease (1, 2). The basic steps in translation have been mapped by genetic, biochemical, structural, and mechanistic studies. However, how translation is regulated and subverted, for example, during viral infection, remains poorly understood, especially in eukaryotes. All viruses compete for the cellular translation machinery to synthesize viral proteins required for virus proliferation. To that end, many viruses contain a structured internal ribosome entry site (IRES) in the 5′ untranslated region of their genome, which allows them to bypass the requirement for certain translation initiation factors. How IRESs achieve this goal remains unclear.Recently, it has been shown that structurally and evolutionarily very diverse IRESs, such as hepatitis C virus (HCV) IRES, cricket paralysis virus (CrPV) IRES (3), and others (4), require ribosomal protein eS25 (RPS25) for efficient translation initiation, indicating that RPS25/IRES interactions could be a universal feature of IRES-mediated translation. RPS25 is located on the back of the head of the 40S ribosomal subunit, distal to the mRNA entry channel but proximal to different IRES RNAs as shown by cryo-EM structures of 80S:CrPV IRES and 80S:HCV IRES complexes. RPS25 is not essential for cap-dependent translation, suggesting that IRES/RPS25 interactions are required to bypass the requirement for the full set of initiation factors (3).Translation initiation is a multistep process, with kinetics and dynamic interrogation refractory to conventional biochemical and biophysical methods. Single-molecule approaches provide insight into compositional and conformational dynamics of these asynchronous processes by following individual molecular events in real time. In bacteria, single-molecule methods allow direct observation of the multiple initiation pathways and conformational rearrangements that guide translation initiation (5, 6). The lack of fluorescently labeled components of translation initiation has prevented application of the single-molecule methods to study the mechanism of translation initiation in humans.Here, we demonstrate an approach to create fluorescently labeled 40S ribosomal subunits from human cells. Using these subunits, we measure the kinetics and conformational pathway of 40S subunit recruitment to the HCV IRES. First, we created RPS25 KO cell lines using the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system. Next, we expressed RPS25 as a C-terminal fusion with mutant O⁶-alkylguanine DNA alkyltransferase (SNAP) as the sole source of the protein. We established biochemically that RPS25-SNAP is efficiently incorporated into functional 40S subunits, and rescues the defect in HCV IRES-mediated translation caused by RPS25 deletion. Using single-molecule fluorescence, we showed that Förster resonance energy transfer (FRET) between RPS25 and the base of domain II of the HCV IRES is dynamic. Conformational dynamics were altered by the presence of translational extract, thus indicating that conformational flexibility of RPS25 and domain II positions could be responsible for guiding downstream steps of translation initiation on HCV IRES. Finally, by measuring the kinetics of the 40S:HCV IRES interactions, we demonstrate that the rate of 40S subunit recruitment to the HCV IRES is not limited by the conformational flexibility of the free HCV IRES, and that upon 40S binding, the 40S:HCV IRES complexes are stable on the time scale of initiation. This study demonstrates the power of real-time observations of conformational and compositional dynamics during human translation initiation.     

Hepatitis C viral protein translation: mechanisms and implications in developing antivirals

Hepatitis C viral protein translation occurs in a cap‐independent manner through the use of an internal ribosomal entry site (IRES) present within the viral 5′‐untranslated region. The IRES is composed of highly conserved structural domains that directly recruit the 40S ribosomal subunit to the viral genomic RNA. This frees the virus from relying on a large number of translation initiation factors that are required for cap‐dependent translation, conferring a selective advantage to the virus especially in times when the availability of such factors is low. Although the mechanism of translation initiation on the Hepatitis C virus (HCV) IRES is well established, modulation of the HCV IRES activity by both cellular and viral factors is not well understood. As the IRES is essential in the HCV life cycle and as such remains well conserved in an otherwise highly heterogenic virus, the process of HCV protein translation represents an attractive target in the development of novel antivirals. This review will focus on the mechanisms of HCV protein translation and how this process is postulated to be modulated by cis‐acting viral factors, as well as trans‐acting viral and cellular factors. Numerous therapeutic approaches investigated in targeting HCV protein translation for the development of novel antivirals will also be discussed.     

Selection of cyclic peptide aptamers to HCV IRES RNA using mRNA display

The hepatitis C virus (HCV) is a positive strand RNA flavivirus that is a major causative agent of serious liver disease, making new treatment modalities an urgent priority. Because HCV translation initiation occurs by a mechanism that is fundamentally distinct from that of host mRNAs, it is an attractive target for drug discovery. The translation of HCV mRNA is initiated from an internal ribosomal entry site (IRES), independent of cap and poly(A) recognition and bypassing eIF4F complex formation. We used mRNA display selection technology combined with a simple and robust cyclization procedure to screen a peptide library of >10(13) different sequences and isolate cyclic peptides that bind with high affinity and specificity to HCV IRES RNA. The best peptide binds the IRES with subnanomolar affinity, and a specificity of at least 100-fold relative to binding to several other RNAs of similar length. The peptide specifically inhibits HCV IRES-initiated translation in vitro with no detectable effect on normal cap-dependent translation initiation. An 8-aa cyclic peptide retains most of the activity of the full-length 27-aa bicyclic peptide. These peptides may be useful tools for the study of HCV translation and may have potential for further development as an anti-HCV drug.     

Mass spectrometry reveals modularity and a complete subunit interaction map of the eukaryotic translation factor eIF3

The eukaryotic initiation factor 3 (eIF3) plays an important role in translation initiation, acting as a docking site for several eIFs that assemble on the 40S ribosomal subunit. Here, we use mass spectrometry to probe the subunit interactions within the human eIF3 complex. Our results show that the 13-subunit complex can be maintained intact in the gas phase, enabling us to establish unambiguously its stoichiometry and its overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Dissociation takes place as a function of ionic strength to form three stable modules eIF3(c:d:e:l:k), eIF3(f:h:m), and eIF3(a:b:i:g). These modules are linked by interactions between subunits eIF3b:c and eIF3c:h. We confirmed our interaction map with the homologous yeast eIF3 complex that contains the five core subunits found in the human eIF3 and supplemented our data with results from immunoprecipitation. These results, together with the 27 subcomplexes identified with increasing ionic strength, enable us to define a comprehensive interaction map for this 800-kDa species. Our interaction map allows comparison of free eIF3 with that bound to the hepatitis C virus internal ribosome entry site (HCV-IRES) RNA. We also compare our eIF3 interaction map with related complexes, containing evolutionarily conserved protein domains, and reveal the location of subunits containing RNA recognition motifs proximal to the decoding center of the 40S subunit of the ribosome.     

Direct functional interaction of initiation factor eIF4G with type 1 internal ribosomal entry sites

Viral internal ribosomal entry sites (IRESs) mediate end-independent translation initiation. There are 4 major structurally-distinct IRES groups: type 1 (e.g., poliovirus) and type 2 (e.g., encephalomyocarditis virus), which are dissimilar except for a Yn-Xm-AUG motif at their 3′ borders, type 3 (e.g., hepatitis C virus), and type 4 (dicistroviruses). Type 2–4 IRESs mediate initiation by distinct mechanisms that are nevertheless all based on specific noncanonical interactions with canonical components of the translation apparatus, such as eukaryotic initiation factor (eIF) 4G (type 2), 40S ribosomal subunits (types 3 and 4), and eIF3 (type 3). The mechanism of initiation on type 1 IRESs is unknown. We now report that domain V of type 1 IRESs, which is adjacent to the Yn-Xm-AUG motif, specifically interacts with the central domain of eIF4G. The position and orientation of eIF4G relative to the Yn-Xm-AUG motif is analogous in type 1 and 2 IRESs. eIF4G promotes recruitment of eIF4A to type 1 IRESs, and together, eIF4G and eIF4A induce conformational changes at their 3′ borders. The ability of mutant type 1 IRESs to bind eIF4G/eIF4A correlated with their translational activity. These characteristics parallel the mechanism of initiation on type 2 IRESs, in which the key event is binding of eIF4G to the J–K domain adjacent to the Yn-Xm-AUG motif, which is enhanced by eIF4A. These data suggest that fundamental aspects of the mechanisms of initiation on these unrelated classes of IRESs are similar.     

Base pairing between hepatitis C virus RNA and 18S rRNA is required for IRES-dependent translation initiation in vivo

Degeneracy in eukaryotic translation initiation is evident in the initiation strategies of various viruses. Hepatitis C virus (HCV) provides an exceptional example—translation of the HCV RNA is facilitated by an internal ribosome entry site (IRES) that can autonomously bind a 40S ribosomal subunit and accurately position it at the initiation codon. This binding involves both ribosomal protein and 18S ribosomal RNA (rRNA) interactions. In this study, we evaluate the functional significance of the rRNA interaction and show that HCV IRES activity requires a 3-nt Watson–Crick base-pairing interaction between the apical loop of subdomain IIId in the IRES and helix 26 in 18S rRNA. Mutations of these nucleotides in either RNA dramatically disrupted IRES activity. The activities of the mutated HCV IRESs could be restored by compensatory mutations in the 18S rRNA. The effects of the 18S rRNA mutations appeared to be specific inasmuch as ribosomes containing these mutations did not support translation mediated by the wild-type HCV IRES, but did not block translation mediated by the cap structure or other viral IRESs. The present study provides, to our knowledge, the first functional demonstration of mRNA–rRNA base pairing in mammalian cells. By contrast with other rRNA-binding sites in mRNAs that can enhance translation as independent elements, e.g., the Shine–Dalgarno sequence in prokaryotes, the rRNA-binding site in the HCV IRES functions as an essential component of a more complex interaction.HCV is a single-stranded RNA virus that is a major cause of severe liver disease. The RNA genome contains a large ORF and expresses a single polypeptide that is processed into smaller proteins, which are necessary for replication and assembly of viral particles. The RNA genome is uncapped, and translation does not require the eukaryotic initiation factor 4F (eIF4F) complex (1), which mediates cap-dependent translation. Instead, translation is facilitated by an internal ribosome entry site (IRES) located in the 5′ nontranslated region (2, 3). Inasmuch as the production of all HCV proteins requires the HCV IRES, the HCV IRES is a therapeutic target (4).IRESs encompass a variety of initiation mechanisms and have been extensively studied in viruses, which often exploit the translation capabilities of the host for their own use (5–7). In many cases, the IRES mechanism complements the infection strategy of the virus. In the case of the HCV IRES and other IRESs of this type, the IRES can effectively recruit the host translation machinery by directly binding to 40S ribosomal subunits (1, 8–12). The HCV–ribosome interaction has been shown to require an interaction with ribosomal protein S25 (13) and can also involve the eIF3 complex, which increases the stability of the interaction (14). In the presence of ternary complex, which contains the initiator Met-tRNA, a preinitiation complex can assemble at the start site.Various studies have investigated the binding of the HCV IRES to 40S subunits and identified interactions with several ribosomal proteins by cross-linking (1, 15–17). Interaction via 18S rRNA was first highlighted in a cryo-EM study of the IRES complexed with 80S ribosomes where the apical loop of subdomain IIId of the IRES (Fig. 1A) was found to make contact with the apical loop of helix 26 in 18S rRNA (18) (Fig. S1A).Open in a separate windowFig. 1.Recombinant 18S rRNA supports HCV IRES-dependent translation. (A) Schematic representation of the secondary structure of the HCV IRES (modified from ref. 26 with permission from Oxford University Press). The location and sequence of domain IIId (nucleotides 266–268) is indicated; the position of U228 at the junction connecting subdomains a–c is shown by the arrow. The sequence of subdomain IIId is shown, and the three nucleotides that bind to 18S rRNA helix 26 are indicated in red bold type. (B) Reporter assays of N2a cells coexpressing recombinant mouse 18S rRNA (wild-type) and a dicistronic reporter construct. The reporter constructs contain the wild-type HCV IRES (HCV WT) or mutations in domain IIId of the IRES (HCV M1–M4), as indicated. The 5′-3′ convention for writing nucleotide sequences is used throughout the figures where sequences are not explicitly labeled. The negative control contains an MCS. A control mutation, U228C (HCV M5), is located at another site in the HCV IRES (see A). The results are reported as IRES activity by normalizing Pluc activity with Rluc activity. Error bars show SD from three independent experiments. An asterisk indicates significance between HCV WT and each of the other constructs (P value < 0.01 in two-sample t tests).More recently, Hashem et al. (12) observed this interaction at higher resolution and suggested that it is probably mediated by base pairing between nucleotides ²⁶⁶GGG²⁶⁸ of subdomain IIId (HCV subtype 1b numbering) and ¹¹¹⁸CCC¹¹²⁰ of helix 26 in 18S rRNA (mouse numbering). This IRES-rRNA interaction is supported by studies showing that mutations in the HCV IRES at nucleotides ²⁶⁶GGG²⁶⁸, which are predicted to disrupt base pairing to 18S rRNA, drastically reduced the binding affinity of the IRES to 40S subunits (8, 19). These mutations also disrupted IRES activity, both in vitro and in cells (19–23). In addition, when complexed with 40S subunits, the IIId loop of the HCV IRES was protected from cleavage by RNase T1 (8, 24) or from modification by kethoxal (25). Moreover, the HCV IRES protects the region ¹¹¹⁵AUUCCC¹¹²⁰ of helix 26 in 18S rRNA from hydroxyl radical cleavage and ¹¹¹⁸CCC¹¹²⁰ from dimethyl sulfate modification (26).Although a strong indication for the intermolecular interaction between HCV IRES and 18S rRNA has been provided by various studies (see above), they are largely limited to cell-free experiments. Other studies that used equivalent or the same methodologies, however, failed to observe the interactions; e.g., see refs. 16, 27, and 28. This discrepancy may be due in part to different conditions or materials used in the experiments. Verifying a putative base-pairing interaction requires demonstrating that activity is disrupted by mutations that disrupt base-pairing potential, and is restored by compensatory mutations in the other RNA that restore pairing potential. It is only with evidence of this type that the functional relevance of a putative base-pairing interaction can be determined. However, until recently, it has not been possible to directly test the predicted pairing interaction as it has not been possible to analyze mutated 18S rRNAs in mammalian cells. Here, we test the predicted base-pairing interaction using a synthetic 18S rRNA expression system that we have developed in mouse cells (29). This system recapitulates the functionality of the native 18S rRNA, including the ability to support translation of exogenous genes.     

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.     

The helicase protein DHX29 promotes translation initiation,cell proliferation,and tumorigenesis

Translational control plays an important role in cell growth and tumorigenesis. Cap-dependent translation initiation of mammalian mRNAs with structured 5′UTRs requires the DExH-box protein, DHX29, in vitro. Here we show that DHX29 is important for translation in vivo. Down-regulation of DHX29 leads to impaired translation, resulting in disassembly of polysomes and accumulation of mRNA-free 80S monomers. DHX29 depletion also impedes cancer cell growth in culture and in xenografts. Thus, DHX29 is a bona fide translation initiation factor that potentially can be exploited as a target to inhibit cancer cell growth.Initiation is a tightly regulated rate-limiting step in the translation of eukaryotic mRNAs. Ribosome recruitment to the mRNA commences with binding of translation initiation factor 4F (eIF4F) to the 7-methyl guanosine cap structure, which is present at the 5′ end of all nuclear-encoded eukaryotic mRNAs (1). eIF4F (comprising the cap-binding protein eIF4E, the DEAD-box RNA helicase eIF4A and eIF4G, a scaffold for binding eIF4E and eIF4A) binds to the cap, unwinds (with the aid of eIF4A) the cap-proximal region of the mRNA, and, through interaction with the ribosome-bound eIF3, recruits the 40S ribosomal subunit to the mRNA (2–4). The 40S subunit then scans the 5′ UTR in a 5′ to 3′ direction until it encounters an initiation codon. A subsequent joining of the 60S ribosomal subunit and release of eIFs result in formation of an elongation-competent 80S ribosome.Secondary structures in 5′UTRs of mRNAs are thought to become unwound to allow ribosomal complexes to move along the mRNA in search of the initiation codon. Thus, in addition to its role in the initial attachment of ribosomal complexes to mRNA, eIF4A is believed to assist ribosomal complexes during scanning (5). Recent observations suggest that the process of eukaryotic initiation requires additional members of the DEAD/DExH-box protein family; for instance, a DEAD-box protein, yeast Ded1, and its mammalian homologue, DDX3, are biochemically and genetically implicated in translation initiation on long structured 5′UTRs (6), and another DExH-box protein, DHX29, strongly stimulates cap-dependent initiation on mRNAs with structured 5′UTRs in vitro (7). Here we studied the importance of DHX29 for translation in vivo and characterized it as a novel factor required for cell proliferation.     

Internal initiation in Saccharomyces cerevisiae mediated by an initiator tRNA/eIF2-independent internal ribosome entry site element

Internal initiation of translation can be mediated by specific internal ribosome entry site (IRES) elements that are located in certain mammalian and viral mRNA molecules. Thus far, these mammalian cellular and viral IRES elements have not been shown to function in the yeast Saccharomyces cerevisiae. We report here that a recently discovered IRES located in the genome of cricket paralysis virus can direct the efficient translation of a second URA3 cistron in dicistronic mRNAs in S. cerevisiae, thereby conferring uracil-independent growth. Curiously, the IRES functions poorly in wild-type yeast but functions efficiently either in the presence of constitutive expression of the eIF2 kinase GCN2 or in cells that have two initiator tRNA(met) genes disrupted. Both of these conditions have been shown to lower the amounts of ternary eIF2-GTP/initiator tRNA(met) complexes. Furthermore, tRNA(met)-independent initiation was also observed in translation-competent extracts prepared from S. cerevisiae in the presence of edeine, a compound that has been shown to interfere with start codon recognition by ribosomal subunits carrying ternary complexes. Therefore, the cricket paralysis virus IRES is likely to recruit ribosomes by internal initiation in S. cerevisiae in the absence of eIF2 and initiator tRNA(met), by the same mechanism of factor-independent ribosome recruitment used in mammalian cells. These findings will allow the use of yeast genetics to determine the mechanism of internal ribosome entry.     

Translational control of protein synthesis in pancreatic acinar cells

Translational control of protein synthesis in the pancreas is important in regulating growth and the synthesis of digestive enzymes. Regulation of translation is primarily directed at the steps in initiation and involves reversible phosphorylation of initiation factors (eIFs) and ribosomal proteins. Major sites include the assembly of the eIF4F mRNA cap binding complex, the activity of guanine nucleotide exchange factor eIF2B, and the activity of ribosomal S6 kinase. All of these involve phosphorylation by different regulatory pathways. Stimulation of protein synthesis in acinar cells is primarily mediated by the phosphatidylinositol 3-kinase-mTOR pathway and involves both release of eIF4E (the limiting component of eIF4F) from its binding protein and phosphorylation of ribosomal S6 protein by S6K. eIF4E is itself phosphorylated by a distinct pathway. Inhibition of acinar protein synthesis can be mediated by inhibition of eIF2B following phosphorylation of eIF2α.     

Poly(A) leader of eukaryotic mRNA bypasses the dependence of translation on initiation factors

Eukaryotic mRNAs in which a poly(A) sequence precedes the initiation codon are known to exhibit a significantly enhanced cap-independent translation, both in vivo and in cell-free translation systems. Consistent with high expression levels of poxviral mRNAs, they contain poly(A) sequences at their 5′ ends, immediately before the initiation AUG codon. Here we show that poly(A) as a leader sequence in mRNA constructs promotes the recruitment of the 40S ribosomal subunits and the efficient formation of initiation complexes at cognate AUG initiation codons in the absence of two essential translation initiation factors, eIF3 and eIF4F. These factors are known to be indispensable for the cap-dependent (and ATP-dependent) mechanism of translation initiation but are shown here to be not required if an mRNA contains a 5′-proximal poly(A). Thus, the presence of a pre-AUG poly(A) sequence results in an alternative mechanism of translation initiation. It involves the binding of initiating 40S ribosomal subunits within the 5′ UTR and their phaseless, ATP-independent, diffusional movement (“phaseless wandering”) along the leader sequence, with subsequent recognition of the initiation (AUG) codon.     

Taura syndrome virus IRES initiates translation by binding its tRNA-mRNA–like structural element in the ribosomal decoding center

In cap-dependent translation initiation, the open reading frame (ORF) of mRNA is established by the placement of the AUG start codon and initiator tRNA in the ribosomal peptidyl (P) site. Internal ribosome entry sites (IRESs) promote translation of mRNAs in a cap-independent manner. We report two structures of the ribosome-bound Taura syndrome virus (TSV) IRES belonging to the family of Dicistroviridae intergenic IRESs. Intersubunit rotational states differ in these structures, suggesting that ribosome dynamics play a role in IRES translocation. Pseudoknot I of the IRES occupies the ribosomal decoding center at the aminoacyl (A) site in a manner resembling that of the tRNA anticodon-mRNA codon. The structures reveal that the TSV IRES initiates translation by a previously unseen mechanism, which is conceptually distinct from initiator tRNA-dependent mechanisms. Specifically, the ORF of the IRES-driven mRNA is established by the placement of the preceding tRNA-mRNA–like structure in the A site, whereas the 40S P site remains unoccupied during this initial step.Protein synthesis relies on precise placement of the ORF within the ribosome during translation initiation. Canonical initiation in eukaryotes depends on a 7-methylguanosine cap at the 5′ terminus of mRNA and on extraribosomal initiation factors (1). Following a stepwise assembly, the 80S initiation complex contains the initiator methionyl-tRNA^Met and the AUG start codon in the peptidyl (P) site. Some viral mRNAs use alternative cap-independent mechanisms that involve internal ribosome entry sites (IRESs) (2). IRESs are folded RNA structures in the 5′ UTR that promote formation of the 80S initiation complex in the presence of fewer initiation factors than required for cap-dependent initiation (3).The ribosomal P-site employment in initiation is thought to be ubiquitous for cap-dependent and IRES-dependent translation (4). Of the four groups of known IRESs, the most streamlined mechanism has been described for IRESs from the Dicistroviridae family of arthropod-infecting viruses. The Dicistroviridae genome has two ORFs separated by an intergenic region (IGR). The IGR contains an IRES that drives translation of the second ORF without the aid of initiation factors (4). Based on phylogenetic analyses of the structural polyprotein ORF2 and IGR IRES, the Dicistroviridae viruses are divided into the genus Cripavirus [including cricket paralysis virus (CrPV), Drosophila C virus, and Plautia stali intestine virus (PSIV)] and Aparavirus [including Taura syndrome virus (TSV), Kashmir bee virus, and acute bee paralysis virus] (4). Biochemical studies suggest that despite differences between some secondary structure elements of Cripavirus and Aparavirus IRESs, the molecular mechanisms of translation initiation are similar (5). IGR IRESs can initiate translation on ribosomes from yeast, wheat, human, and other eukaryotic organisms, indicating that the molecular mechanism of IGR IRES-driven initiation in eukaryotes is conserved and is not species-specific (6–10).In contrast to cap-dependent initiation and initiation from other groups of IRESs, translation from IGR IRESs starts from a non-AUG start codon and does not involve initiator methionyl-tRNA^Met. Translation from the majority of IGR IRESs, including the CrPV and TSV IRESs, initiates with alanyl-tRNA^Ala (7, 9, 10). IGR IRESs contain three pseudoknots. At the 5′ region, pseudoknot II (PKII) and PKIII, which are critical for formation of the 40S•IRES and 80S•IRES complexes (8, 9), form a double-nested pseudoknot (11, 12). PKI, located immediately upstream of the start codon, forms a separate domain at the 3′ region of the IRES. This domain is essential for the function of IGR IRESs (13). The crystal structure of an isolated PKI of the CrPV IGR IRES shows that the pseudoknot resembles the anticodon stem loop of tRNA bound to a cognate mRNA codon (14, 15). Isolated PKI of CrPV and PSIV IRESs binds to the P site of the bacterial 70S ribosome, demonstrating that PKI has an affinity to the highly conserved tRNA binding sites on the ribosome (16).The molecular mechanism of translation initiation by IGR IRESs is not fully understood. The current view is that upon formation of the 80S•IRES complex, the PKI is placed in the P site on the small subunit, in a manner mimicking the initiator methionyl-tRNA^Met and the AUG codon (6–8, 10, 17). In this mode, the IRES would position the ORF on the ribosome by presenting the initiating alanine codon in the A (aminoacyl) site. The structural studies of the mechanism, however, have been inconclusive. Previous electron cryomicroscopy (cryo-EM) reconstruction of the CrPV IRES bound to human ribosomal 40S subunit revealed the IRES density spanning from the A site to beyond the exit (E) site (18). The interpretation of the 40S•IRES map favored a model in which PKI interacts with the P-site region (18), although the 20-Å map lacked detailed features in this location. Cryo-EM studies of the 80S ribosome-bound CrPV IRES suggested that upon subunit joining and 80S•IRES complex formation, the IRES may rearrange relative to the 40S subunit (18), and/or reposition PKI in the vicinity of the A and P sites, yet potentially present the downstream alanine codon in the A site (19). The density for the PKI region in these 20-Å and 7.3-Å cryo-EM reconstructions was, however, significantly weaker than that for the rest of the CrPV IRES (18, 19), and it remained unclear how the IGR IRESs initiate translation by accurately positioning the ORF on the 80S ribosome. We report here ∼6-Å cryo-EM structures of the initiation 80S complex bound with an intergenic IRES, which provide structural insights into the mechanism of IGR IRES-driven initiation.     

Initiation factor 2, tRNA, and 50S subunits cooperatively stabilize mRNAs on the ribosome during initiation

Initiation factor 2 (IF2) is a key factor in initiation of bacterial protein synthesis. It recruits initiator tRNA to the small ribosomal subunit and facilitates joining of the large ribosomal subunit. Using reconstituted translation system of Escherichia coli and optical tweezers, we directly measure the rupture force between single ribosomal complexes and mRNAs for initiation complexes in the presence and the absence of IF2. We demonstrate that IF2 together with codon recognition by initiator tRNA increases the force required to dislocate mRNA from the ribosome complexes; mRNA stabilization by IF2 required the presence of a joined 50S subunit, and was independent of bound guanine nucleotide. IF2 thus helps lock the 70S ribosome over the start codon during initiation, thus maintaining reading frame. Our results show how mRNA is progressively stabilized on the ribosome through distinct steps of initiation.     

Crystal structure of the yeast eIF4A-eIF4G complex: an RNA-helicase controlled by protein-protein interactions

Translation initiation factors eIF4A and eIF4G form, together with the cap-binding factor eIF4E, the eIF4F complex, which is crucial for recruiting the small ribosomal subunit to the mRNA 5' end and for subsequent scanning and searching for the start codon. eIF4A is an ATP-dependent RNA helicase whose activity is stimulated by binding to eIF4G. We report here the structure of the complex formed by yeast eIF4G's middle domain and full-length eIF4A at 2.6-A resolution. eIF4A shows an extended conformation where eIF4G holds its crucial DEAD-box sequence motifs in a productive conformation, thus explaining the stimulation of eIF4A's activity. A hitherto undescribed interaction involves the amino acid Trp-579 of eIF4G. Mutation to alanine results in decreased binding to eIF4A and a temperature-sensitive phenotype of yeast cells that carry a Trp579Ala mutation as its sole source for eIF4G. Conformational changes between eIF4A's closed and open state provide a model for its RNA-helicase activity.     

Initiation context modulates autoregulation of eukaryotic translation initiation factor 1 (eIF1)

The central feature of standard eukaryotic translation initiation is small ribosome subunit loading at the 5' cap followed by its 5' to 3' scanning for a start codon. The preferred start is an AUG codon in an optimal context. Elaborate cellular machinery exists to ensure the fidelity of start codon selection. Eukaryotic initiation factor 1 (eIF1) plays a central role in this process. Here we show that the translation of eIF1 homologs in eukaryotes from diverse taxa involves initiation from an AUG codon in a poor context. Using human eIF1 as a model, we show that this poor context is necessary for an autoregulatory negative feedback loop in which a high level of eIF1 inhibits its own translation, establishing that variability in the stringency of start codon selection is used for gene regulation in eukaryotes. We show that the stringency of start codon selection (preferential utilization of optimal start sites) is increased to a surprising degree by overexpressing eIF1. The capacity for the cellular level of eIF1 to impact initiation through the variable stringency of initiation codon selection likely has significant consequences for the proteome in eukaryotes.