Communication between eukaryotic translation initiation factors 1 and 1A on the yeast small ribosomal subunit

We have used expressed protein ligation to site-specifically label eukaryotic translation initiation factors (eIFs) 1 and 1A at their C termini with tetramethyl rhodamine. These fluorescent proteins were used in steady-state anisotropy-based binding experiments to measure the dissociation constants of the factors and the yeast small (40S) ribosomal subunit for the first time. These studies demonstrate that both eIF1 and eIF1A are capable of binding to the 40S subunit in the absence of any other initiation factors or mRNA, arguing against previous suggestions that eIF3 is required for recruitment of eIF1 to the small ribosomal subunit. Strikingly, the data also demonstrate that there is approximately ninefold thermodynamic coupling in the binding of the two factors to the 40S subunit. This indicates that eIF1 and eIF1A communicate with one another when bound to the 40S subunit. Communication between these two factors is likely to be important for coordinating their functions during the initiation process. The data presented here provide a foundation on which to build a quantitative understanding of the network of interactions between these essential factors and the rest of the initiation machinery.     

Small ribosomal protein RPS0 stimulates translation initiation by mediating 40S-binding of eIF3 via its direct contact with the eIF3a/TIF32 subunit

The ribosome translates information encoded by mRNAs into proteins in all living cells. In eukaryotes, its small subunit together with a number of eukaryotic initiation factors (eIFs) is responsible for locating the mRNA's translational start to properly decode the genetic message that it carries. This multistep process requires timely and spatially coordinated placement of eIFs on the ribosomal surface. In our long-standing pursuit to map the 40S-binding site of one of the functionally most complex eIFs, yeast multisubunit eIF3, we identified several interactions that placed its major body to the head, beak and shoulder regions of the solvent-exposed side of the 40S subunit. Among them is the interaction between the N-terminal domain (NTD) of the a/TIF32 subunit of eIF3 and the small ribosomal protein RPS0A, residing near the mRNA exit channel. Previously, we demonstrated that the N-terminal truncation of 200 residues in tif32-Δ8 significantly reduced association of eIF3 and other eIFs with 40S ribosomes in vivo and severely impaired translation reinitiation that eIF3 ensures. Here we show that not the first but the next 200 residues of a/TIF32 specifically interact with RPS0A via its extreme C-terminal tail (CTT). Detailed analysis of the RPS0A conditional depletion mutant revealed a marked drop in the polysome to monosome ratio suggesting that the initiation rates of cells grown under non-permissive conditions were significantly impaired. Indeed, amounts of eIF3 and other eIFs associated with 40S subunits in the pre-initiation complexes in the RPS0A-depleted cells were found reduced; consistently, to the similar extent as in the tif32-Δ8 cells. Similar but less pronounced effects were also observed with the viable CTT-less mutant of RPS0A. Together we conclude that the interaction between the flexible RPS0A-CTT and the residues 200-400 of the a/TIF32-NTD significantly stimulates attachment of eIF3 and its associated eIFs to small ribosomal subunits in vivo.     

Dual function of eIF3j/Hcr1p in processing 20 S pre-rRNA and translation initiation

eIF3j/Hcr1p, a protein associated with eIF3, was shown to bind to, and stabilize, the multifactor complex containing eIFs 1, 2, 3, and 5 and Met-tRNA(i)(Met), whose formation is required for an optimal rate of translation initiation. Here we present evidence that eIF3j/Hcr1p is an RNA binding protein that enhances a late step in 40 S ribosome maturation involving cleavage of the 20 S precursor of 18 S rRNA in the cytoplasm. Immunofluorescence staining shows that eIF3j/Hcr1p is localized predominantly in the cytoplasm. The hcr1Delta mutant exhibits a decreased amount of 40 S subunits, hypersensitivity to paromomycin, and increased levels of 20 S pre-rRNA. Combining the hcr1Delta mutation with drs2Delta or rps0aDelta, deletions of two other genes involved in the same step of 40 S subunit biogenesis, produced a synthetic growth defect. p35, the human ortholog of eIF3j/Hcr1p, partially complemented the slow growth phenotype conferred by hcr1Delta when overexpressed in yeast. heIF3j/p35 was found physically associated with yeast eIF3 and 43 S initiation complexes in vitro and in vivo. Because it did not complement the 40 S biogenesis defect of hcr1Delta, it appears that heIF3j can substitute for eIF3j/Hcr1p only in translation initiation. We conclude that eIF3j/Hcr1p is required for rapid processing of 20 S to 18 S rRNA besides its role in translation initiation, providing an intriguing link between ribosome biogenesis and translation.     

eIF4B controls survival and proliferation and is regulated by proto-oncogenic signaling pathways

Messenger RNA translation, or protein synthesis, is a fundamental biological process affecting cell growth, survival and proliferation. Initiation is the rate limiting and hence the most regulated step of translation. In eukaryotes, translation initiation is facilitated by multiple protein factors collectively called eIFs (for eukaryotic translation initiation factors). The complex consisting of the eIF4 group factors including the mRNA cap-binding eIF4E protein, large scaffolding protein eIF4G and RNA helicase eIF4A is assisted by the eIF4B co-factor to unwind local secondary structures and create a ribosome landing pad on mRNA. Recruitment of the ribosome and augmentation in the mRNA scanning process culminates in the positioning of the ribosome over the start codon. Deregulated translational control is believed to play an important role in oncogenic transformation. Indeed, many eIFs are bona fide proto-oncogenes. In many types of human cancers, eIFs are either overexpressed or ectopically activated by Ras-MAPK and PI3K-mTOR signaling cascades, resulting in increased survival and accelerated proliferation. In this review we will analyze the bulk of data describing eIF4B and its role in cell survival and proliferation. Recent studies have shown that eIF4B is phosphorylated and activated by Ras-MAPK and PI3K-mTOR signaling cascades. In addition, eIF4B regulates translation of proliferative and pro-survival mRNAs. Moreover, eIF4B depletion in cancer cells attenuates proliferation, sensitizes them to genotoxic stress driven apoptosis. Taken together, these findings identify eIF4B as a potential target for development of anti-cancer therapies.     

Position of the CrPV IRES on the 40S subunit and factor dependence of IRES/80S ribosome assembly

The cricket paralysis virus intergenic region internal ribosomal entry site (CrPV IGR IRES) can assemble translation initiation complexes by binding to 40S subunits without Met-tRNA(Met)(i) and initiation factors (eIFs) and then by joining directly with 60S subunits, yielding elongation-competent 80S ribosomes. Here, we report that eIF1, eIF1A and eIF3 do not significantly influence IRES/40S subunit binding but strongly inhibit subunit joining and the first elongation cycle. The IRES can avoid their inhibitory effect by its ability to bind directly to 80S ribosomes. The IRES's ability to bind to 40S subunits simultaneously with eIF1 allowed us to use directed hydroxyl radical cleavage to map its position relative to the known position of eIF1. A connecting loop in the IRES's pseudoknot (PK) III domain, part of PK II and the entire domain containing PK I are solvent-exposed and occupy the E site and regions of the P site that are usually occupied by Met-tRNA(Met)(i).     

eIF4B controls survival and proliferation and is regulated by proto-oncogenic signaling pathways

Messenger RNA translation or protein synthesis, is a fundamental biological process affecting cell growth, survival and proliferation. Initiation is the rate limiting and hence the most regulated step of translation. In eukaryotes, translation initiation is facilitated by multiple protein factors collectively called eIFs (for eukaryotic translation initiation factors). The complex consisting of the eIF4 group factors including the mRNA cap-binding eIF4E protein, large scaffolding protein eIF4G and RNA helicase eIF4A is assisted by the eIF4B co-factor to unwind local secondary structures and create a ribosome landing pad on mRNA. Recruitment of the ribosome and augmentation in the mRNA scanning process culminates in the positioning of the ribosome over the start codon. Deregulated translational control is believed to play an important role in oncogenic transformation. Indeed, many eIFs are bona fide proto-oncogenes. In many types of human cancers, eIFs are either overexpressed or ectopically activated by Ras-MAPK and PI3K-mTOR signaling cascades, resulting in increased survival and accelerated proliferation. In this review we will analyze the bulk of data describing eIF4B and its role in cell survival and proliferation. Recent studies have shown that eIF4B is phosphorylated and activated by Ras-MAPK and PI3K-mTOR signaling cascades. In addition, eIF4B regulates translation of proliferative and pro-survival mRNAs. Moreover, eIF4B depletion in cancer cells attenuates proliferation, sensitizes them to genotoxic stress-driven apoptosis. Taken together, these findings identify eIF4B as a potential target for development of anti-cancer therapies.Key words: eIF4B, translation, signaling, structured 5′UTR, helicase activity, survival, proliferation, apoptosis     

Minor secondary-structure variation in the 5'-untranslated region of the beta-globin mRNA changes the concentration requirements for eIF2

Nucleotide sequence changes increasing the number of paired bases without producing stable secondary structure elements in the 5'-untranslated region (5'-UTR) of the beta-globin mRNA had a slight effect on its translation in rabbit reticulocyte lysate at its low concentration and dramatically decreased translation efficiency at a high concentration. The removal of paired regions restored translation. Addition of purified eIF2 to the lysate resulted in equal translation efficiencies of templates differing in structure of 5'-UTR. A similar effect was observed for p50, a major mRNP protein. Other mRNA-binding initiation factors, eIF4F and eIF3B, had no effect on the dependence of translation efficiency on mRNA concentration. Analysis of the assembly of the 48S initiation complex from its purified components showed that less eIF2 is required for translation initiation on the beta-globin mRNA than on its derivative containing minor secondary structure elements in 5'-UTR. According to a model proposed, eIF2 not only delivers Met-tRNA, but it also stabilizes the complex of the 40S ribosome subunit with 5'-UTR, which is of particular importance for translation initiation on templates with structured 5'-UTR.     

Translation Initiation Factor eIF4B Interacts with a Picornavirus Internal Ribosome Entry Site in both 48S and 80S Initiation Complexes Independently of Initiator AUG Location

Most eukaryotic initiation factors (eIFs) are required for internal translation initiation at the internal ribosome entry site (IRES) of picornaviruses. eIF4B is incorporated into ribosomal 48S initiation complexes with the IRES RNA of foot-and-mouth disease virus (FMDV). In contrast to the weak interaction of eIF4B with capped cellular mRNAs and its release upon entry of the ribosomal 60S subunit, eIF4B remains tightly associated with the FMDV IRES during formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. The interaction of eIF4B with the IRES in 48S and 80S complexes is independent of the location of the initiator AUG and thus independent of the mechanism by which the small ribosomal subunit is placed at the actual start codon, either by direct internal ribosomal entry or by scanning. eIF4B does not greatly rearrange its binding to the IRES upon entry of the ribosomal subunits, and the interaction of eIF4B with the IRES is independent of the polypyrimidine tract-binding protein, which enhances FMDV translation.     

Phosphorylation of human eukaryotic initiation factor 2γ: novel site identification and targeted PKC involvement

Eukaryotic translation requires a suite of proteins known as eukaryotic initiation factors (eIFs). These molecular effectors oversee the highly regulated initiation phase of translation. Essential to eukaryotic translation initiation is the protein eIF2, a heterotrimeric protein composed of the individually distinct subunits eIF2α, eIF2β, and eIF2γ. The ternary complex, formed when eIF2 binds to GTP and Met-tRNA(i), is responsible for shuttling Met-tRNA(i) onto the awaiting 40S ribosome. As a necessary component for translation initiation, much attention has been given to the phosphorylation of eIF2α. Despite several previous investigations into eIF2 phosphorylation, most have centered on α- or β-subunit phosphorylation and little is known regarding γ-subunit phosphorylation. Herein, we report eight sites of phosphorylation on the largest eIF2 subunit with seven novel phosphosite identifications via high resolution mass spectrometry. Of the eight sites identified, three are located in either the switch regions or nucleotide binding pocket domain. In addition, we have identified a possible kinase of eIF2, protein kinase C (PKC), which is capable of phosphorylating threonine 66 (thr-66) on the intact heterotrimer. These findings may shed new light on the regulation of ternary complex formation and alternate molecular effectors involved in this process prior to 80S ribosome formation and subsequent translation elongation and termination.     

Mammalian stress granules represent sites of accumulation of stalled translation initiation complexes

In eukaryotic cells subjected to environmental stress, untranslated mRNA accumulates in discrete cytoplasmic foci that have been termed stress granules. Recent studies have shown that in addition to mRNA, stress granules also contain 40S ribosomal subunits and various translation initiation factors, including the mRNA binding proteins eIF4E and eIF4G. However, eIF2, the protein that transfers initiator methionyl-tRNA(i) (Met-tRNA(i)) to the 40S ribosomal subunit, has not been detected in stress granules. This result is surprising because the eIF2. GTP. Met-tRNA(i) complex is thought to bind to the 40S ribosomal subunit before the eIF4G. eIF4E. mRNA complex. In the present study, we show in both NIH-3T3 cells and mouse embryo fibroblasts that stress granules contain not only eIF2 but also the guanine nucleotide exchange factor for eIF2, eIF2B. Moreover, we show that phosphorylation of the alpha-subunit of eIF2 is necessary and sufficient for stress granule formation during the unfolded protein response. Finally, we also show that stress granules contain many, if not all, of the components of the 48S preinitiation complex, but not 60S ribosomal subunits, suggesting that they represent stalled translation initiation complexes.     

Coupled release of eukaryotic translation initiation factors 5B and 1A from 80S ribosomes following subunit joining

The translation initiation GTPase eukaryotic translation initiation factor 5B (eIF5B) binds to the factor eIF1A and catalyzes ribosomal subunit joining in vitro. We show that rapid depletion of eIF5B in Saccharomyces cerevisiae results in the accumulation of eIF1A and mRNA on 40S subunits in vivo, consistent with a defect in subunit joining. Substituting Ala for the last five residues in eIF1A (eIF1A-5A) impairs eIF5B binding to eIF1A in cell extracts and to 40S complexes in vivo. Consistently, overexpression of eIF5B suppresses the growth and translation initiation defects in yeast expressing eIF1A-5A, indicating that eIF1A helps recruit eIF5B to the 40S subunit prior to subunit joining. The GTPase-deficient eIF5B-T439A mutant accumulated on 80S complexes in vivo and was retained along with eIF1A on 80S complexes formed in vitro. Likewise, eIF5B and eIF1A remained associated with 80S complexes formed in the presence of nonhydrolyzable GDPNP, whereas these factors were released from the 80S complexes in assays containing GTP. We propose that eIF1A facilitates the binding of eIF5B to the 40S subunit to promote subunit joining. Following 80S complex formation, GTP hydrolysis by eIF5B enables the release of both eIF5B and eIF1A, and the ribosome enters the elongation phase of protein synthesis.     

Poly(A)-binding protein facilitates translation of an uncapped/nonpolyadenylated viral RNA by binding to the 3' untranslated region

Viruses employ an alternative translation mechanism to exploit cellular resources at the expense of host mRNAs and to allow preferential translation. Plant RNA viruses often lack both a 5' cap and a 3' poly(A) tail in their genomic RNAs. Instead, cap-independent translation enhancer elements (CITEs) located in the 3' untranslated region (UTR) mediate their translation. Although eukaryotic translation initiation factors (eIFs) or ribosomes have been shown to bind to the 3'CITEs, our knowledge is still limited for the mechanism, especially for cellular factors. Here, we searched for cellular factors that stimulate the 3'CITE-mediated translation of Red clover necrotic mosaic virus (RCNMV) RNA1 using RNA aptamer-based one-step affinity chromatography, followed by mass spectrometry analysis. We identified the poly(A)-binding protein (PABP) as one of the key players in the 3'CITE-mediated translation of RCNMV RNA1. We found that PABP binds to an A-rich sequence (ARS) in the viral 3' UTR. The ARS is conserved among dianthoviruses. Mutagenesis and a tethering assay revealed that the PABP-ARS interaction stimulates 3'CITE-mediated translation of RCNMV RNA1. We also found that both the ARS and 3'CITE are important for the recruitment of the plant eIF4F and eIFiso4F factors to the 3' UTR and of the 40S ribosomal subunit to the viral mRNA. Our results suggest that dianthoviruses have evolved the ARS and 3'CITE as substitutes for the 3' poly(A) tail and the 5' cap of eukaryotic mRNAs for the efficient recruitment of eIFs, PABP, and ribosomes to the uncapped/nonpolyadenylated viral mRNA.     

Regulation of translation initiation by insulin and amino acids in skeletal muscle of neonatal pigs

Previous studies have shown that intravenous infusion of insulin and/or amino acids reproduces the feeding-induced stimulation of muscle protein synthesis in neonates and that insulin and amino acids act independently to produce this effect. The goal of the present study was to delineate the regulatory roles of insulin and amino acids on muscle protein synthesis in neonates by examining translational control mechanisms, specifically the eukaryotic translation initiation factors (eIFs), which enable coupling of initiator methionyl-tRNAi and mRNA to the 40S ribosomal subunit. Insulin secretion was blocked by somatostatin in fasted 7-day-old pigs (n = 8-12/group), insulin was infused to achieve plasma levels of approximately 0, 2, 6, and 30 microU/ml, and amino acids were clamped at fasting or fed levels or, at the high insulin dose, below fasting. Both insulin and amino acids increased the phosphorylation of ribosomal protein S6 kinase (S6K1) and the eIF4E-binding protein (4E-BP1), decreased the binding of 4E-BP1 to eIF4E, increased eIF4E binding to eIF4G, and increased fractional protein synthesis rates but did not affect eIF2B activity. In the absence of insulin, amino acids had no effect on these translation initiation factors but increased the protein synthesis rates. Raising insulin from below fasting to fasting levels generally did not alter translation initiation factor activity but raised protein synthesis rates. The phosphorylation of S6K1 and 4E-BP1 and the amount of 4E-BP1 bound to eIF4E and eIF4E bound to eIF4G were correlated with insulin level, amino acid level, and protein synthesis rate. Thus insulin and amino acids regulate muscle protein synthesis in skeletal muscle of neonates by modulating the availability of eIF4E for 48S ribosomal complex assembly, although other processes also must be involved.     

Eukaryotic translation initiation factor 3 (eIF3) and eIF2 can promote mRNA binding to 40S subunits independently of eIF4G in yeast

Recruitment of the eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNAiMet ternary complex to the 40S ribosome is stimulated by multiple initiation factors in vitro, including eIF3, eIF1, eIF5, and eIF1A. Recruitment of mRNA is thought to require the functions of eIF4F and eIF3, with the latter serving as an adaptor between the ribosome and the 4G subunit of eIF4F. To define the factor requirements for these reactions in vivo, we examined the effects of depleting eIF2, eIF3, eIF5, or eIF4G in Saccharomyces cerevisiae cells on binding of the ternary complex, other initiation factors, and RPL41A mRNA to native 43S and 48S preinitiation complexes. Depleting eIF2, eIF3, or eIF5 reduced 40S binding of all constituents of the multifactor complex (MFC), comprised of these three factors and eIF1, supporting a mechanism of coupled 40S binding by MFC components. 40S-bound mRNA strongly accumulated in eIF5-depleted cells, even though MFC binding to 40S subunits was reduced by eIF5 depletion. Hence, stimulation of the GTPase activity of the ternary complex, a prerequisite for 60S subunit joining in vitro, is likely the rate-limiting function of eIF5 in vivo. Depleting eIF2 or eIF3 impaired mRNA binding to free 40S subunits, but depleting eIF4G led unexpectedly to accumulation of mRNA on 40S subunits. Thus, it appears that eIF3 and eIF2 are more critically required than eIF4G for stable binding of at least some mRNAs to native preinitiation complexes and that eIF4G has a rate-limiting function at a step downstream of 48S complex assembly in vivo.     

EIF3 p170, a mediator of mimosine effect on protein synthesis and cell cycle progression

l-Mimosine, a plant amino acid, can reversibly block mammalian cells at late G1 phase and has been suggested to affect translation of mRNAs such as p27, the CDK inhibitor. However, the mechanism of this effect is not known. Regulation of translation generally occurs at the initiation step that, in mammalian cells, is a complex process that requires multiple eukaryotic initiation factors (eIFs) and ribosome. The effects of mimosine on initiation factors or regulators consequently will influence translation initiation. P170, a putative subunit of eIF3, has been suggested to be nonessential for eIF3 function to form preinitiation complexes and it may function as a regulator for translation of a subset of mRNAs. In this article, we tested this hypothesis and investigated whether eIF3 p170 mediates mimosine effect on mRNA translation. We found that p170 translation was dramatically reduced by mimosine due to its iron-chelating function. The decreased expression of p170 by mimosine caused diminished de novo synthesis of tyrosinated alpha-tubulin and elevated translation of p27 before cell cycle arrest. These observations suggest that p170 is likely an early response gene to mimosine treatment and a mediator for mimosine effect on mRNA translation. The effect of p170 on the synthesis of tyrosinated alpha-tubulin and p27 in a reciprocal manner also suggests that p170 functions as a regulator for mRNA translation.     

A region rich in aspartic acid, arginine, tyrosine, and glycine (DRYG) mediates eukaryotic initiation factor 4B (eIF4B) self-association and interaction with eIF3.

The binding of mRNA to the ribosome is mediated by eukaryotic initiation factors eukaryotic initiation factor 4F (eIF4F), eIF4B, eIF4A, and eIF3, eIF4F binds to the mRNA cap structure and, in combination with eIF4B, is believed to unwind the secondary structure in the 5' untranslated region to facilitate ribosome binding. eIF3 associates with the 40S ribosomal subunit prior to mRNA binding. eIF4B copurifies with eIF3 and eIF4F through several purification steps, suggesting the involvement of a multisubunit complex during translation initiation. To understand the mechanism by which eIF4B promotes 40S ribosome binding to the mRNA, we studied its interactions with partner proteins by using a filter overlay (protein-protein [far Western]) assay and the two-hybrid system. In this report, we show that eIF4B self-associates and also interacts directly with the p170 subunit of eIF3. A region rich in aspartic acid, arginine, tyrosine, and glycine, termed the DRYG domain, is sufficient for self-association of eIF4B, both in vitro and in vivo, and for interaction with the p170 subunit of eIF3. These experiments suggest that eIF4B participates in mRNA-ribosome binding by acting as an intermediary between the mRNA and eIF3, via a direct interaction with the p170 subunit of eIF3.     

mRNA helicases: the tacticians of translational control

The translation initiation step in eukaryotes is highly regulated and rate-limiting. During this process, the 40S ribosomal subunit is usually recruited to the 5' terminus of the mRNA. It then migrates towards the initiation codon, where it is joined by the 60S ribosomal subunit to form the 80S initiation complex. Secondary structures in the 5' untranslated region (UTR) can impede binding and movement of the 40S ribosome. The canonical eukaryotic translation initiation factor eIF4A (also known as DDX2), together with its accessory proteins eIF4B and eIF4H, is thought to act as a helicase that unwinds secondary structures in the mRNA 5' UTR. Growing evidence suggests that other helicases are also important for translation initiation and may promote the scanning processivity of the 40S subunit, synergize with eIF4A to 'melt' secondary structures or facilitate translation of a subset of mRNAs.     

eIF3j is located in the decoding center of the human 40S ribosomal subunit

Protein synthesis in all cells begins with the ordered binding of the small ribosomal subunit to messenger RNA (mRNA) and transfer RNA (tRNA). In eukaryotes, translation initiation factor 3 (eIF3) is thought to play an essential role in this process by influencing mRNA and tRNA binding through indirect interactions on the backside of the 40S subunit. Here we show by directed hydroxyl radical probing that the human eIF3 subunit eIF3j binds to the aminoacyl (A) site and mRNA entry channel of the 40S subunit, placing eIF3j directly in the ribosomal decoding center. eIF3j also interacts with eIF1A and reduces 40S subunit affinity for mRNA. A high affinity for mRNA is restored upon recruitment of initiator tRNA, even though eIF3j remains in the mRNA-binding cleft in the presence of tRNA. These results suggest that eIF3j functions in part by regulating access of the mRNA-binding cleft in response to initiation factor binding.     

Cloning and characterization of the p42 subunit of mammalian translation initiation factor 3 (eIF3): demonstration that eIF3 interacts with eIF5 in mammalian cells.

Eukaryotic translation initiation factor 3 (eIF3) is a large multisubunit protein complex that plays an essential role in the binding of the initiator methionyl-tRNA and mRNA to the 40S ribosomal subunit to form the 40S initiation complex. cDNAs encoding all the subunits of mammalian eIF3 except the p42 subunit have been cloned in several laboratories. Here we report the cloning and characterization of a human cDNA encoding the p42 subunit of mammalian eIF3. The open reading frame of the cDNA, which encodes a protein of 320 amino acids (calculated Mr35 614) has been expressed in Escherichia coli and the recombinant protein has been purified to homogeneity. The purified protein binds RNA in agreement with the presence of a putative RNA binding motif in the deduced amino acid sequence. The protein shows 33% identity and 53% similarity with the Tif35p subunit (YDR 429C) of yeast eIF3. Transfection experiments demonstrated that polyhistidine-tagged p42 protein, transiently expressed in human U20S cells, was incorporated into endogenous eIF3. Furthermore, eIF3 isolated from transfected cell lysates contains bound eIF5 indicating that a specific physical interaction between eIF5 and eIF3 may play an important role in the function of eIF5 during translation initiation in eukaryotic cells.