Characterization of eIF3k: a newly discovered subunit of mammalian translation initiation factor elF3.

Mammalian translation initiation factor 3 (eIF3) is a multisubunit complex containing at least 12 subunits with an apparent aggregate mass of approximately 700 kDa. eIF3 binds to the 40S ribosomal subunit, promotes the binding of methionyl-tRNAi and mRNA, and interacts with several other initiation factors to form the 40S initiation complex. Human cDNAs encoding 11 of the 12 subunits have been isolated previously; here we report the cloning and characterization of a twelfth subunit, a 28-kDa protein named eIF3k. Evidence that eIF3k is present in the eIF3 complex was obtained. A monoclonal anti-eIF3a (p170) Ig coimmunoprecipitates eIF3k with the eIF3 complex. Affinity purification of histidine-tagged eIF3k from transiently transfected COS cells copurifies other eIF3 subunits. eIF3k colocalizes with eIF3 on 40S ribosomal subunits. eIF3k coexpressed with five other 'core' eIF3 subunits in baculovirus-infected insect cells, forms a stable, immunoprecipitatable, complex with the 'core'. eIF3k interacts directly with eIF3c, eIF3g and eIF3j by glutathione S-transferase pull-down assays. Sequences homologous with eIF3k are found in the genomes of Caenorhabitis elegans, Arabidopsis thaliana and Drosophila melanogaster, and a homologous protein has been reported to be present in wheat eIF3. Its ubiquitous expression in human tissues, yet its apparent absence in Saccharomyces cerevisiae and Schizosaccharomyces pombe, suggest a unique regulatory role for eIF3k in higher organisms. The studies of eIF3k complete the characterization of mammalian eIF3 subunits.     

The eIF3 complex of Leishmania—subunit composition and mode of recruitment to different cap-binding complexes

Eukaryotic initiation factor 3 (eIF3) is a multi-protein complex and a key participant in the assembly of the translation initiation machinery. In mammals, eIF3 comprises 13 subunits, most of which are characterized by conserved structural domains. The trypanosomatid eIF3 subunits are poorly conserved. Here, we identify 12 subunits that comprise the Leishmania eIF3 complex (LeishIF3a-l) by combining bioinformatics with affinity purification and mass spectrometry analyses. These results highlight the strong association of LeishIF3 with LeishIF1, LeishIF2 and LeishIF5, suggesting the existence of a multi-factor complex. In trypanosomatids, the translation machinery is tightly regulated in the different life stages of these organisms as part of their adaptation and survival in changing environments. We, therefore, addressed the mechanism by which LeishIF3 is recruited to different mRNA cap-binding complexes. A direct interaction was observed in vitro between the fully assembled LeishIF3 complex and recombinant LeishIF4G3, the canonical scaffolding protein of the cap-binding complex in Leishmania promastigotes. We further highlight a novel interaction between the C-terminus of LeishIF3a and LeishIF4E1, the only cap-binding protein that efficiently binds the cap structure under heat shock conditions, anchoring a complex that is deficient of any MIF4G-based scaffolding subunit.     

The translation initiation complex eIF3 in trypanosomatids and other pathogenic excavates – identification of conserved and divergent features based on orthologue analysis

Background

The initiation of translation in eukaryotes is supported by the action of several eukaryotic Initiation Factors (eIFs). The largest of these is eIF3, comprising of up to thirteen polypeptides (eIF3a through eIF3m), involved in multiple stages of the initiation process. eIF3 has been better characterized from model organisms, but is poorly known from more diverged groups, including unicellular lineages represented by known human pathogens. These include the trypanosomatids (Trypanosoma and Leishmania) and other protists belonging to the taxonomic supergroup Excavata (Trichomonas and Giardia sp.).

Results

An in depth bioinformatic search was carried out to recover the full content of eIF3 subunits from the available genomes of L. major, T. brucei, T. vaginalis and G. duodenalis. The protein sequences recovered were then submitted to homology analysis and alignments comparing them with orthologues from representative eukaryotes. Eleven putative eIF3 subunits were found from both trypanosomatids whilst only five and four subunits were identified from T. vaginalis and G. duodenalis, respectively. Only three subunits were found in all eukaryotes investigated, eIF3b, eIF3c and eIF3i. The single subunit found to have a related Archaean homologue was eIF3i, the most conserved of the eIF3 subunits. The sequence alignments revealed several strongly conserved residues/region within various eIF3 subunits of possible functional relevance. Subsequent biochemical characterization of the Leishmania eIF3 complex validated the bioinformatic search and yielded a twelfth eIF3 subunit in trypanosomatids, eIF3f (the single unidentified subunit in trypanosomatids was then eIF3m). The biochemical data indicates a lack of association of the eIF3j subunit to the complex whilst highlighting the strong interaction between eIF3 and eIF1.

Conclusions

The presence of most eIF3 subunits in trypanosomatids is consistent with an early evolution of a fully functional complex. Simplified versions in other excavates might indicate a primordial complex or secondary loss of selected subunits, as seen for some fungal lineages. The conservation in eIF3i sequence might indicate critical functions within eIF3 which have been overlooked. The identification of eIF3 subunits from distantly related eukaryotes provides then a basis for the study of conserved/divergent aspects of eIF3 function, leading to a better understanding of eukaryotic translation initiation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1175) contains supplementary material, which is available to authorized users.     

eIF3k inhibits NF-κB signaling by targeting MyD88 for ATG5-mediated autophagic degradation in teleost fish

Optimal activation of NF-κB signaling is crucial for the initiation of inflammatory responses and eliminating invading bacteria. Bacteria have likewise evolved the ability to evade immunity; however, mechanisms by which bacteria dysregulate host NF-κB signaling are unclear. In this study, we identify eukaryotic translation initiation factor eIF3k, a nonessential member of the eIF3 translation initiation complex, as a suppressor of the NF-κB pathway. Mechanistically, we show that eIF3k expression induced by Vibrio harveyi enhances E3 ligase Nrdp1-mediated K27-linked ubiquitination of MyD88, an upstream regulator of NF-κB pathway activation. Furthermore, we show that eIF3k acts as a bridge linking ubiquitin-tagged MyD88 and ATG5, an important mediator of autophagy. We demonstrate that the MyD88-eIF3k-ATG5 complex is transported to the autophagosome for degradation, and that innate immune signaling is subsequently terminated and does not attack invading V. harveyi. Therefore, our study identifies eIF3k as a specific inhibitor of the MyD88-dependent NF-κB pathway and suggests that eIF3k may act as a selective autophagic receptor that synergizes with ATG5 to promote the autophagic degradation of MyD88, which helps V. harveyi to evade innate immunity. We conclude that V. harveyi can manipulate a host''s autophagy process to evade immunity in fish and also provide a new perspective on mammalian resistance to bacterial invasion.     

Expression,purification, and crystallization of <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> eIF2B

Tight control of protein synthesis is necessary for cells to respond and adapt to environmental changes rapidly. Eukaryotic translation initiation factor (eIF) 2B, the guanine nucleotide exchange factor for eIF2, is a key target of translation control at the initiation step. The nucleotide exchange activity of eIF2B is inhibited by the stress-induced phosphorylation of eIF2. As a result, the level of active GTP-bound eIF2 is lowered, and protein synthesis is attenuated. eIF2B is a large multi-subunit complex composed of five different subunits, and all five of the subunits are the gene products responsible for the neurodegenerative disease, leukoencephalopathy with vanishing white matter. However, the overall structure of eIF2B has remained unresolved, due to the difficulty in preparing a sufficient amount of the eIF2B complex. To overcome this problem, we established the recombinant expression and purification method for eIF2B from the fission yeast Schizosaccharomyces pombe. All five of the eIF2B subunits were co-expressed and reconstructed into the complex in Escherichia coli cells. The complex was successfully purified with a high yield. This recombinant eIF2B complex contains each subunit in an equimolar ratio, and the size exclusion chromatography analysis suggests it forms a heterodecamer, consistent with recent reports. This eIF2B increased protein synthesis in the reconstituted in vitro human translation system. In addition, disease-linked mutations led to subunit dissociation. Furthermore, we crystallized this functional recombinant eIF2B, and the crystals diffracted to 3.0 Å resolution.     

The Saccharomyces cerevisiae Homologue of Mammalian Translation Initiation Factor 6 Does Not Function as a Translation Initiation Factor

Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. The Saccharomyces cerevisiae gene that encodes the 245-amino-acid eIF6 (calculated M_r 25,550), designated TIF6, has been cloned and expressed in Escherichia coli. The purified recombinant protein prevents association between 40S and 60S ribosomal subunits to form 80S ribosomes. TIF6 is a single-copy gene that maps on chromosome XVI and is essential for cell growth. eIF6 expressed in yeast cells associates with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Depletion of eIF6 from yeast cells resulted in a decrease in the rate of protein synthesis, accumulation of half-mer polyribosomes, reduced levels of 60S ribosomal subunits resulting in the stoichiometric imbalance in the 40S/60S subunit ratio, and ultimately cessation of cell growth. Furthermore, lysates of yeast cells depleted of eIF6 remained active in translation of mRNAs in vitro. These results indicate that eIF6 does not act as a true translation initiation factor. Rather, the protein may be involved in the biogenesis and/or stability of 60S ribosomal subunits.     

The m Subunit of Murine Translation Initiation Factor eIF3 Maintains the Integrity of the eIF3 Complex and Is Required for Embryonic Development,Homeostasis, and Organ Size Control

Mammalian eIF3 is composed of 13 subunits and is the largest eukaryotic initiation factor. eIF3 plays a key role in protein biosynthesis. However, it is not fully understood how different subunits contribute to the structural integrity and function of the eIF3 complex. Whether eIF3 is essential for embryonic development and homeostasis is also not known. Here, we show that eIF3m null embryos are lethal at the peri-implantation stage. Compound heterozygotes (eIF3m^flox/−) or FABP4-Cre-mediated conditional knock-out mice are lethal at mid-gestation stages. Although the heterozygotes are viable, they show markedly reduced organ size and diminished body weight. Acute ablation of eIF3m in adult mouse liver leads to rapidly decreased body weight and death within 2 weeks; these effects are correlated with a severe decline of protein biogenesis in the liver. Protein analyses reveal that eIF3m deficiency significantly impairs the integrity of the eIF3 complex due to down-regulation of multiple other subunits. Two of the subunits, eIF3f and eIF3h, are stabilized by eIF3m through subcomplex formation. Therefore, eIF3m is required for the structural integrity and translation initiation function of eIF3. Furthermore, not only is eIF3m an essential gene, but its expression level is also important for mouse embryonic development and the control of organ size.     

Translation Initiation Requires Cell Division Cycle 123 (Cdc123) to Facilitate Biogenesis of the Eukaryotic Initiation Factor 2 (eIF2)

The eukaryotic translation initiation factor 2 (eIF2) is central to the onset of protein synthesis and its modulation in response to physiological demands. eIF2, a heterotrimeric G-protein, is activated by guanine nucleotide exchange to deliver the initiator methionyl-tRNA to the ribosome. Here we report that assembly of the eIF2 complex in vivo depends on Cdc123, a cell proliferation protein conserved among eukaryotes. Mutations of CDC123 in budding yeast reduced the association of eIF2 subunits, diminished polysome levels, and increased GCN4 expression indicating that Cdc123 is critical for eIF2 activity. Cdc123 bound the unassembled eIF2γ subunit, but not the eIF2 complex, and the C-terminal domain III region of eIF2γ was both necessary and sufficient for Cdc123 binding. Alterations of the binding site revealed a strict correlation between Cdc123 binding, the biological function of eIF2γ, and its ability to assemble with eIF2α and eIF2β. Interestingly, high levels of Cdc123 neutralized the assembly defect and restored the biological function of an eIF2γ mutant. Moreover, the combined overexpression of eIF2 subunits rescued an otherwise inviable cdc123 deletion mutant. Thus, Cdc123 is a specific eIF2 assembly factor indispensable for the onset of protein synthesis. Human Cdc123 is encoded by a disease risk locus, and, therefore, eIF2 biogenesis control by Cdc123 may prove relevant for normal cell physiology and human health. This work identifies a novel step in the eukaryotic translation initiation pathway and assigns a biochemical function to a protein that is essential for growth and viability of eukaryotic cells.     

Recruitment of the 40S Ribosome Subunit to the 3′-Untranslated Region (UTR) of a Viral mRNA,via the eIF4 Complex,Facilitates Cap-independent Translation

Barley yellow dwarf virus mRNA, which lacks both cap and poly(A) tail, has a translation element (3′-BTE) in its 3′-UTR essential for efficient translation initiation at the 5′-proximal AUG. This mechanism requires eukaryotic initiation factor 4G (eIF4G), subunit of heterodimer eIF4F (plant eIF4F lacks eIF4A), and 3′-BTE-5′-UTR interaction. Using fluorescence anisotropy, SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) analysis, and toeprinting, we found that (i) 40S subunits bind to BTE (K_d = 350 ± 30 nm), (ii) the helicase complex eIF4F-eIF4A-eIF4B-ATP increases 40S subunit binding (K_d = 120 ± 10 nm) to the conserved stem-loop I of the 3′-BTE by exposing more unpaired bases, and (iii) long distance base pairing transfers this complex to the 5′-end of the mRNA, where translation initiates. Although 3′-5′ interactions have been recognized as important in mRNA translation, barley yellow dwarf virus employs a novel mechanism utilizing the 3′-UTR as the primary site of ribosome recruitment.     

Mouse p56 blocks a distinct function of eukaryotic initiation factor 3 in translation initiation

Members of the p56 family of mammalian proteins are strongly induced in virus-infected cells and in cells treated with interferons or double-stranded RNA. Previously, we have reported that human p56 inhibits initiation of translation by binding to the "e" subunit of eukaryotic initiation factor 3 (eIF3) and subsequently interfering with the eIF3/eIF2.GTP.Met-tRNAi (ternary complex) interaction. Here we report that mouse p56 also interferes with eIF3 functions and inhibits translation. However, the murine protein binds to the "c" subunit, not the "e" subunit, of eIF3. Consequently, it has only a marginal effect on eIF3.ternary complex interaction. Instead, the major inhibitory effect of mouse p56 is manifested at a different step of translation initiation, namely the binding of eIF4F to the 40 S ribosomal subunit.eIF3.ternary complex. Thus, mouse and human p56 proteins block different functions of eIF3 by binding to its different subunits.     

Caspase-mediated cleavage and DNase activity of the translation initiation factor 3, subunit G (eIF3g)

Eukaryotic translation initiation factor 3 is composed of 13 subunits (eIF3a through eIF3m) and plays an essential role in translation. During apoptosis, several caspases rapidly down-regulate protein synthesis by cleaving eIF4G, -4B, -3j, and -2α. In this study, we found that the activation of caspases by cisplatin in T24 cells induces the cleavage of subunit G of the eIF3 complex (eIF3g). The cleavage site (SLRD²²⁰G) was identified, and we found that the cleaved N-terminus was translocated to the nucleus, activating caspase-3, and that it also showed a strong DNase activity. These data demonstrate the important roles of eIF3g in the translation initiation machinery and in DNA degradation during apoptosis.     

The translational regulator eIF3a: The tricky eIF3 subunit!

Regulation of gene expression is a fundamental step in cellular physiology as abnormalities in this process may lead to de-regulated growth and cancer. Translation of mRNA is mainly regulated at the rate-limiting initiation step, where many eukaryotic initiation factors (eIFs) are involved. The largest and most complex initiation factor is eIF3 which plays a role in translational regulation, cell growth and cancer. The largest subunit of eIF3 is eIF3a, although it is not required for the general function of eIF3 in translation initiation. However, eIF3a may play a role as a regulator of a subset of mRNAs and has been demonstrated to regulate the expression of p27^kip1, tyrosinated α-tubulin and ribonucleotide reductase M2 subunit. These molecules have a pivotal role in the regulation of the cell cycle. Moreover, the eIF3a mRNA is ubiquitously expressed in all tissues at different levels and is found elevated in a number of cancer types. eIF3a can modulate the cell cycle and may be a translational regulator for proteins important for entrance into S phase. The expression of eIF3a is decreased in differentiated cells in culture and the suppression of eIF3a expression can reverse the malignant phenotype and change the sensitivity of cells to cell cycle modulators. However, the role of eIF3a in cancer is still unclear. In fact, some studies have identified eIF3a to be involved in cancer development, while other results indicate that it could provide protection against evolution into higher malignancy. Together, these findings highlight the “tricky” and interesting nature of eIF3a.     

Identification of a Translation Initiation Factor 3 (eIF3) Core Complex, Conserved in Yeast and Mammals, That Interacts with eIF5

Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have recognizable homologs encoded in the Saccharomyces cerevisiae genome, and only two of these (Prt1p and Tif34p) were identified previously as subunits of yeast eIF3. We purified a polyhistidine-tagged form of Prt1p (His-Prt1p) by Ni²⁺ affinity and gel filtration chromatography and obtained a complex of ≈600 kDa composed of six polypeptides whose copurification was completely dependent on the polyhistidine tag on His-Prt1p. All five polypeptides associated with His-Prt1p were identified by mass spectrometry, and four were found to be the other putative homologs of human eIF3 subunits encoded in S. cerevisiae: YBR079c/Tif32p, Nip1p, Tif34p, and YDR429c/Tif35p. The fifth Prt1p-associated protein was eIF5, an initiation factor not previously known to interact with eIF3. The purified complex could rescue Met-tRNA_i^Met binding to 40S ribosomes in defective extracts from a prt1 mutant or extracts from which Nip1p had been depleted, indicating that it possesses a known biochemical activity of eIF3. These findings suggest that Tif32p, Nip1p, Prt1p, Tif34p, and Tif35p comprise an eIF3 core complex, conserved between yeast and mammals, that stably interacts with eIF5. Nip1p bound to eIF5 in yeast two-hybrid and in vitro protein binding assays. Interestingly, Sui1p also interacts with Nip1p, and both eIF5 and Sui1p have been implicated in accurate recognition of the AUG start codon. Thus, eIF5 and Sui1p may be recruited to the 40S ribosomes through physical interactions with the Nip1p subunit of eIF3.     

Reconstitution reveals the functional core of mammalian eIF3

Eukaryotic translation initiation factor (eIF)3 is the largest eIF ( approximately 650 kDa), consisting of 10-13 different polypeptide subunits in mammalian cells. To understand the role of each subunit, we successfully reconstituted a human eIF3 complex consisting of 11 subunits that promoted the recruitment of the 40S ribosomal subunit to mRNA. Strikingly, the eIF3g and eIF3i subunits, which are evolutionarily conserved between human and the yeast Saccharomyces cerevisiae are dispensable for active mammalian eIF3 complex formation. Extensive deletion analyses suggest that three evolutionarily conserved subunits (eIF3a, eIF3b, and eIF3c) and three non-conserved subunits (eIF3e, eIF3f, and eIF3h) comprise the functional core of mammalian eIF3.     

Changes in ribosomal binding activity of eIF3 correlate with increased translation rates during activation of T lymphocytes

The rate of protein synthesis in quiescent peripheral blood T lymphocytes increases dramatically following mitogenic activation. The stimulation of translation is due to an increase in the rate of initiation caused by the regulation of initiation factor activities. Here, we focus on eIF3, a large multiprotein complex that plays a central role in the formation of the 40 S initiation complex. Using sucrose density gradient centrifugation to analyze ribosome complexes, we find that most eIF3 is not bound to 40 S ribosomal subunits in unactivated T lymphocytes but becomes ribosome-bound following activation. Immunoblot analyses of sucrose gradient fractions for individual eIF3 subunits show that the small eIF3j subunit is unassociated with the eIF3 complex in quiescent T lymphocytes, but upon activation joins the other eIF3 subunits and binds 40 S ribosomal subunits. Because eIF3j has been shown to be required for eIF3 binding to 40 S ribosomes in vitro, the results suggest that mitogenic stimulation of T lymphocytes leads to an activation of eIF3j, thereby enabling eIF3 to bind to the larger ribosome-free eIF3 subunit complex, and then to the 40 S ribosomes. The association of eIF3j with the other eIF3 subunits appears to be inhibited by rapamycin, suggesting a mechanism that lies downstream from the mammalian target of rapamycin kinase. This association requires ionomycin together with a phorbol ester, which also suggests that calcium signaling is involved. We conclude that the complex formation of eIF3 and its association with the ribosomes might contribute to increased translation rates during T lymphocyte activation.     

Crystal structure of human eIF3k, the first structure of eIF3 subunits

eIF3k, the smallest subunit of eukaryotic initiation factor 3 (eIF3), interacts with several other subunits of eIF3 and the 40 S ribosomal subunit. eIF3k is conserved among high eukaryotes, including mammals, insects, and plants, and it is ubiquitously expressed in human tissues. Interestingly, eIF3k does not exist in some species of yeast. Thus, eIF3k may play a unique regulatory role in higher organisms. Here we report the crystal structure of human eIF3k, the first high-resolution structure of an eIF3 component. This novel structure contains two distinct domains, a HEAT (named for Huntington, elongation factor 3, A subunit of protein phosphatase 2A, target of rapamycin) repeat-like HAM (HEAT analogous motif) domain and a winged-helix-like WH domain. Through structural comparison and sequence conservation analysis, we show that eIF3k has three putative protein-binding surfaces and has potential RNA binding activity. The structure provides key information for understanding the structure and function of the eIF3 complex.     

Structural characterization of the human eukaryotic initiation factor 3 protein complex by mass spectrometry

Protein synthesis in mammalian cells requires initiation factor eIF3, an approximately 800-kDa protein complex that plays a central role in binding of initiator methionyl-tRNA and mRNA to the 40 S ribosomal subunit to form the 48 S initiation complex. The eIF3 complex also prevents premature association of the 40 and 60 S ribosomal subunits and interacts with other initiation factors involved in start codon selection. The molecular mechanisms by which eIF3 exerts these functions are poorly understood. Since its initial characterization in the 1970s, the exact size, composition, and post-translational modifications of mammalian eIF3 have not been rigorously determined. Two powerful mass spectrometric approaches were used in the present study to determine post-translational modifications that may regulate the activity of eIF3 during the translation initiation process and to characterize the molecular structure of the human eIF3 protein complex purified from HeLa cells. In the first approach, the bottom-up analysis of eIF3 allowed for the identification of a total of 13 protein components (eIF3a-m) with a sequence coverage of approximately 79%. Furthermore 29 phosphorylation sites and several other post-translational modifications were unambiguously identified within the eIF3 complex. The second mass spectrometric approach, involving analysis of intact eIF3, allowed the detection of a complex with each of the 13 subunits present in stoichiometric amounts. Using tandem mass spectrometry four eIF3 subunits (h, i, k, and m) were found to be most easily dissociated and therefore likely to be on the periphery of the complex. It is noteworthy that none of these four subunits were found to be phosphorylated. These data raise interesting questions about the function of phosphorylation as it relates to the core subunits of the complex.     

Interaction between 25S rRNA A Loop and Eukaryotic Translation Initiation Factor 5B Promotes Subunit Joining and Ensures Stringent AUG Selection

In yeast, 25S rRNA makes up the major mass and shape of the 60S ribosomal subunit. During the last step of translation initiation, eukaryotic initiation factor 5B (eIF5B) promotes the 60S subunit joining with the 40S initiation complex (IC). Malfunctional 60S subunits produced by misfolding or mutation may disrupt the 40S IC stalling on the start codon, thereby altering the stringency of initiation. Using several point mutations isolated by random mutagenesis, here we studied the role of 25S rRNA in start codon selection. Three mutations changing bases near the ribosome surface had strong effects, allowing the initiating ribosomes to skip both AUG and non-AUG codons: C2879U and U2408C, altering the A loop and P loop, respectively, of the peptidyl transferase center, and G1735A, mapping near a Eukarya-specific bridge to the 40S subunit. Overexpression of eIF5B specifically suppressed the phenotype caused by C2879U, suggesting functional interaction between eIF5B and the A loop. In vitro reconstitution assays showed that C2879U decreased eIF5B-catalyzed 60S subunit joining with a 40S IC. Thus, eIF5B interaction with the peptidyl transferase center A loop increases the accuracy of initiation by stabilizing the overall conformation of the 80S initiation complex. This study provides an insight into the effect of ribosomal mutations on translation profiles in eukaryotes.     

Distinct Regions of Human eIF3 Are Sufficient for Binding to the HCV IRES and the 40S Ribosomal Subunit

Translation of the hepatitis C virus (HCV) genomic RNA initiates from an internal ribosome entry site (IRES) in its 5′ untranslated region and requires a minimal subset of translation initiation factors to occur, namely eukaryotic initiation factor (eIF) 2 and eIF3. Low-resolution structural information has revealed how the HCV IRES RNA binds human eIF3 and the 40S ribosomal subunit and positions the start codon for initiation. However, the exact nature of the interactions between the HCV IRES RNA and the translational machinery remains unknown. Using limited proteolysis and mass spectrometry, we show that distinct regions of human eIF3 are sufficient for binding to the HCV IRES RNA and the 40S subunit. Notably, the eIF3 subunit eIF3b is protected by HCV IRES RNA binding, yet is exposed in the complex when compared to subunits eIF3e, eIF3f, eIF3h, and eIF3l. Limited proteolysis reveals that eIF3 binding to the 40S ribosomal subunit occurs through many redundant interactions that can compensate for each other. These data suggest how the HCV IRES binds to specific regions of eIF3 to target the translational machinery to the viral genomic RNA and provide a framework for modeling the architecture of intact human eIF3.