Eukaryotic initiation factor 4E

Eukaryotic translation initiation factor 4E (eIF4E) is perhaps best known for its function in the initiation of protein synthesis on capped mRNAs in the cytoplasm. However, recent studies have highlighted that eIF4E has many additional functions, which include the nuclear export of specific mRNAs as well as roles in ageing and the translation of some uncapped viral RNAs. This review aims to update the reader on recent developments, including the potential of eIF4E as a therapeutic target.     

The eukaryotic translation initiation factor 4GI is cleaved by different retroviral proteases

The initiation factor eIF4G plays a central role in the regulation of translation. In picornaviruses, as well as in human immunodeficiency virus type 1 (HIV-1), cleavage of eIF4G by the viral protease leads to inhibition of protein synthesis directed by capped cellular mRNAs. In the present work, cleavage of both eIF4GI and eIF4GII has been analyzed by employing the proteases encoded within the genomes of several members of the family Retroviridae, e.g., Moloney murine leukemia virus (MoMLV), mouse mammary tumor virus, human T-cell leukemia virus type 1, HIV-2, and simian immunodeficiency virus. All of the retroviral proteases examined were able to cleave the initiation factor eIF4GI both in intact cells and in cell-free systems, albeit with different efficiencies. The eIF4GI hydrolysis patterns obtained with HIV-1 and HIV-2 proteases were very similar to each other but rather different from those obtained with MoMLV protease. Both eIF4GI and eIF4GII were cleaved very efficiently by the MoMLV protease. However, eIF4GII was a poor substrate for HIV proteases. Proteolytic cleavage of eIF4G led to a profound inhibition of cap-dependent translation, while protein synthesis driven by mRNAs containing internal ribosome entry site elements remained unaffected or was even stimulated in transfected cells.     

Eukaryotic initiation factor 4B from wheat and Arabidopsis thaliana is a member of a multigene family

Clones of eukaryotic initiation factor (eIF) 4B from wheat and Arabidopsis thaliana were obtained from cDNA and genomic libraries. The exon/intron organization of the genes from wheat and A. thaliana is very similar. The deduced amino acid sequences for the wheat and Arabidopsis eIF4B proteins showed overall similarity to each other, but very little similarity to eIF4B from other eukaryotes. The recombinant form of eIF4B supports polypeptide synthesis in an in vitro translation system and reacts with antibodies to native wheat eIF4B. In contrast to mammalian eIF4B and eIF4A, the combination of wheat eIF4B and eIF4A does not stimulate RNA-dependent ATP hydrolysis activity; however, wheat eIF4B does stimulate eIF4F and eIF4A RNA-dependent ATP hydrolysis activity. Interestingly, eIF4B does not stimulate eIF(iso)4F and eIF4A hydrolysis activity. Gel filtration experiments indicate wheat eIF4B, like its mammalian counterpart, self-associates to form a homodimer.     

Eukaryotic initiation factor 4D, the hypusine-containing protein, is conserved among eukaryotes

When mammalian cells are grown in medium containing [3H]spermidine, a single major tritiated protein identical to eukaryotic initiation factor 4D becomes labeled. This protein contains 1 residue/molecule of tritiated hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine), a rare amino acid which has been found in no other protein. In order to investigate the conservation of this protein, we examined two nonmammalian eukaryotes, the yeast Saccharomyces cerevisiae and the insect Drosophila melanogaster, and the eubacterial prokaryote Escherichia coli for the presence of the hypusine-containing protein. When the eukaryotic cells were grown in the presence of [3H]spermidine, electrophoretic analysis revealed a single labeled protein. In each case, the apparent molecular weight was near 18,000 and the relative pI was approximately 5.2, similar to the hypusine-containing protein of mammals. Amino acid analysis confirmed the presence of tritiated hypusine in each case, and silver staining of two-dimensional polyacrylamide gels demonstrated that, in yeast and fruit flies as in mammals, the protein is relatively abundant. In the eubacterium E. coli, one tritiated protein was predominant, but its molecular weight was 24,000 and we found no evidence that it contained tritiated hypusine. We found no evidence for the existence of the hypusine-containing protein in the archaebacterium Methanococcus voltae. These data suggest that the hypusine-containing protein is conserved among eukaryotes.     

Eukaryotic initiation factor eIF2

eIF2 plays a central role in the maintenance of what is generally considered a rate-limiting step in mRNA translation. In this step, eIF2 binds GTP and Met-tRNAi and transfers Met-tRNAi to the 40S ribosomal subunit. At the end of the initiation process, GTP bound to eIF2 is hydrolyzed to GDP and the eIF2.GDP complex is released from the ribosome. The exchange of GDP bound to eIF2 for GTP is a prerequisite to binding Met-tRNAi and is mediated by a second initiation factor, eIF2B. In what is probably the best-characterized mechanism for the regulation of mRNA translation, phosphorylation of eIF2 on its smallest, or alpha-, subunit converts eIF2 from a substrate of eIF2B into a competitive inhibitor. Thus, phosphorylation of eIF2 alpha effectively prevents formation of the eIF2.GTP.Met-tRNAi complex and inhibits global protein synthesis. Phosphorylation of eIF2 alpha occurs under a variety of conditions including viral infection, apoptosis, nutrient deprivation, heme-deprivation, and certain stresses.     

The p220 component of eukaryotic initiation factor 4F is a substrate for multiple calcium-dependent enzymes

Eukaryotic initiation factor 4F (eIF-4F) is a multisubunit protein that functions in the first step of the binding of capped mRNAs to the small ribosomal subunit. Its largest polypeptide component, p220, is cleaved following poliovirus infection. This is thought to inactivate eIF-4F function, thereby preventing cap-dependent initiation of translation of cellular mRNAs. In this report, we show that p220 in extracts of uninfected HeLa cells is specifically lost in the presence of calcium. The responsible activities have been partially purified and identified as the calcium-dependent, neutral, cysteine proteases calpains I and II. In addition, a third calcium-dependent activity was resolved from the calpains and also results in the loss of p220. This activity has properties similar to a transglutaminase and copurifies with tissue transglutaminase through several chromatographic steps. None of these calcium-dependent activities appears to mediate p220 cleavage in poliovirus-infected cells.     

Eukaryotic translation initiation factor 3 subunit b is a novel oncogenic factor in prostate cancer

Mammalian Genome - Prostate cancer, the second most common cancer among male adults, affects millions globally. We sought to investigate the expression and contribution of Eukaryotic translation...     

Eukaryotic translation initiation factor 4F architectural alterations accompany translation initiation factor redistribution in poxvirus-infected cells

Despite their self-sufficient ability to generate capped mRNAs from cytosolic DNA genomes, poxviruses must commandeer the critical eukaryotic translation initiation factor 4F (eIF4F) to recruit ribosomes. While eIF4F integrates signals to control translation, precisely how poxviruses manipulate the multisubunit eIF4F, composed of the cap-binding eIF4E and the RNA helicase eIF4A assembled onto an eIF4G platform, remains obscure. Here, we establish that the poxvirus infection of normal, primary human cells destroys the translational repressor eIF4E binding protein (4E-BP) and promotes eIF4E assembly into an active eIF4F complex bound to the cellular polyadenylate-binding protein (PABP). Stimulation of the eIF4G-associated kinase Mnk1 promotes eIF4E phosphorylation and enhances viral replication and protein synthesis. Remarkably, these eIF4F architectural alterations are accompanied by the concentration of eIF4E and eIF4G within cytosolic viral replication compartments surrounded by PABP. This demonstrates that poxvirus infection redistributes, assembles, and modifies core and associated components of eIF4F and concentrates them within discrete subcellular compartments. Furthermore, it suggests that the subcellular distribution of eIF4F components may potentiate the complex assembly.     

Eukaryotic translation initiation factor 4E is a cellular target for toxicity and death due to exposure to cadmium chloride

Whether translation initiation factor 4E (eIF4E), the mRNA cap binding and rate-limiting factor required for translation, is a target for cytotoxicity and cell death induced by cadmium, a human carcinogen, was investigated. Exposure of human cell lines, HCT15, PLC/PR/5, HeLa, and Chang, to cadmium chloride resulted in cytotoxicity and cell death, and this was associated with a significant decrease in eIF4E protein levels. Similarly, specific silencing of the expression of the eIF4E gene, caused by a small interfering RNA, resulted in significant cytotoxicity and cell death. On the other hand, overexpression of the eIF4E gene was protective against the cadmium-induced cytotoxicity and cell death. Further studies revealed the absence of alterations in the eIF4E mRNA level in the cadmium-treated cells despite their decreased eIF4E protein level. In addition, exposure of cells to cadmium resulted in enhanced ubiquitination of eIF4E protein while inhibitors of proteasome activity reversed the cadmium-induced decrease of eIF4E protein. Exposure of cells to cadmium, as well as the specific silencing of eIF4E gene, also resulted in decreased cellular levels of cyclin D1, a critical cell cycle and growth regulating gene, suggesting that the observed inhibition of cyclin D1 gene expression in the cadmium-treated cells is most likely due to decreased cellular level of eIF4E. Taken together, our results demonstrate that the exposure of cells to cadmium chloride resulted in cytotoxicity and cell death due to enhanced ubiquitination and consequent proteolysis of eIF4E protein, which in turn diminished cellular levels of critical genes such as cyclin D1.     

Eukaryotic initiation factor (eIF)-4F. Implications for a role in internal initiation of translation

In order to study the eukaryotic translation initiation mechanisms of "internal initiation," "re-initiation," and/or "coupled internal initiation," a series of model mRNAs have been constructed which contain two non-overlapping open reading frames (ORFs) that encode different lengths of rabbit alpha globin. These mRNAs, along with the bicistronic constructs TK/CAT and TK/P2CAT developed by Pelletier and Sonenberg (Pelletier, J., and Sonenberg, N. (1988) Nature 334, 320-325, 1988), were used to program an in vitro rabbit reticulocyte lysate translation system. Cap-dependent and cap-independent translation were distinguished by monitoring translation in the presence or absence of exogenously added cap analog (m7GTP). Messenger RNAs which translate both ORF1 and ORF2 by a cap-dependent mechanism, as well as mRNAs that translate ORF2 by a cap-independent mechanism while still translating ORF1 in a cap-dependent fashion have been obtained. These same alpha globin mRNAs differ by no more than 45 nucleotides in intercistronic length. Initiation factor addition studies were performed in this same in vitro translation system. Both eukaryotic initiation factor (eIF)-4F and, to a lesser extent, eIF-4B can stimulate translation of an internally located ORF independent of upstream ORF translation and in a manner not dependent on mRNA cap recognition. This indicates that the cap-recognition initiation factor, eIF-4F, and eIF-4B facilitate cap-independent and internal initiation of an open reading frame.     

Fibronectin is a non-viral substrate for the HIV proteinase

The retrovirus encoded proteinase (PR) is required for the proper maturation of viral particles into infectious virus. The PR had been considered highly substrate specific, cleaving exclusively the viral gag and gag-pol protein precursor. It has recently been reported, however, that cytoskeleton and other cellular filament proteins can be cleaved by the HIV-1 PR. Here we have evidence that a cell-associated protein, the fibronectin (A-chain), is also cleaved in vitro specifically by this PR. The possibility of a cytotoxic role of the PR is conceivable.     

eIF4A2 is a host factor required for efficient HIV-1 replication

Host factors are required for efficient HIV-1 replication. To identify these factors, genome-wide RNA interference screening was performed using a human T cell line. In the present study, we assessed whether eukaryotic translation initiation factor 4A isoform 2 (eIF4A2), a DEAD-box protein identified in our screen, is necessary for efficient HIV-1 replication. Exploiting MT4C5 cells depleted of eIF4A2 by stable expression of eIF4A2-specific short-hairpin RNA (shRNA) using a lentiviral system, we found that depletion of eIF4A2 markedly inhibited the infection of a replication-competent reporter HIV-1. eIF4A2 depletion reduced the efficiency of viral cDNA synthesis with virion entry into target cells being unaffected. Depletion of eIF4A2 also inhibited HIV-1 spreading infection in a knockdown level-dependent manner. These results suggest that HIV-1 requires eIF4A2 for optimal replication in human T cells.     

Eukaryotic initiation factor 4A stimulates translation in microinjected Xenopus oocytes

The injection of heterologous mRNA into fully grown Xenopus oocytes results not only in the synthesis of the heterologous protein but also in a reciprocal decrease in the synthesis of endogenous proteins. This indicates that injected and endogenous mRNAs compete for some component which is rate-limiting for translation in oocytes. We have attempted to identify this rate-limiting translational component. We find that heterologous and homologous polysomes compete with endogenous mRNAs as effectively as naked mRNA, indicating that polysomes do not contain detectable levels of the rate-limiting factor. In addition, we have used micrococcal nuclease digestion and a mRNA-specific oligonucleotide to destroy the mRNA component of polysomes. The remaining polysome factors, when injected into oocytes, failed to stimulate translation. When several eukaryotic translation initiation factors were injected into oocytes, initiation factor 4A consistently increased general oocyte protein synthesis by about twofold. It is possible that the availability of eIF-4A in oocytes is a key factor in limiting the overall rate of protein synthesis.     

Eukaryotic initiation factor 4E-3 is essential for meiotic chromosome segregation, cytokinesis and male fertility in Drosophila

Gene expression is translationally regulated during many cellular and developmental processes. Translation can be modulated by affecting the recruitment of mRNAs to the ribosome, which involves recognition of the 5' cap structure by the cap-binding protein eIF4E. Drosophila has several genes encoding eIF4E-related proteins, but the biological role of most of them remains unknown. Here, we report that Drosophila eIF4E-3 is required specifically during spermatogenesis. Males lacking eIF4E-3 are sterile, showing defects in meiotic chromosome segregation, cytokinesis, nuclear shaping and individualization. We show that eIF4E-3 physically interacts with both eIF4G and eIF4G-2, the latter being a factor crucial for spermatocyte meiosis. In eIF4E-3 mutant testes, many proteins are present at different levels than in wild type, suggesting widespread effects on translation. Our results imply that eIF4E-3 forms specific eIF4F complexes that are essential for spermatogenesis.     

Eukaryotic translation initiation factor 5 functions as a GTPase-activating protein

Eukaryotic translation initiation factor 5 (eIF5) forms a complex with eIF2 by interacting with the beta subunit of eIF2. This interaction is essential for eIF5-promoted hydrolysis of GTP bound to the 40 S initiation complex. In this work, we show that, in addition to the eIF2 beta-binding region at the C terminus of eIF5, the N-terminal region of eIF5 is also required for eIF5-dependent GTP hydrolysis. Like other GTPase-activating proteins, eIF5 contains an invariant arginine residue (Arg-15) at its N terminus that is essential for its function. Mutation of this arginine residue to alanine or even to conservative lysine caused a severe defect in the ability of eIF5 to promote GTP hydrolysis from the 40 S initiation complex, although the ability of these mutant proteins to bind to eIF2 beta remained unchanged. These mutants were also defective in overall protein synthesis as well as in their ability to support cell growth of a Delta TIF5 yeast strain. Additionally, alanine substitution mutagenesis of eIF5 defined Lys-33 and Lys-55 as also critical for eIF5 function in vitro and in vivo. The implications of these results in relation to other well characterized GAPs are discussed and provide additional evidence that eIF5 functions as a GTPase-activating protein.     

Backbone resonance assignment of the HEAT1-domain of the human eukaryotic translation initiation factor 4GI

Controlling translation during protein synthesis is crucial for cell proliferation and differentiation. Protein translation is orchestrated by an assembly of various protein components at the ribosomal subunits. The eukaryotic translation initiation factor 4G (eIF4G) plays an important role in the formation of the translation initiation complex eIF4F consisting of eIF4G, the ATP dependent RNA helicase eIF4A and the cap binding protein eIF4E. One of the functions of eIF4G is the enhancement of the activity of eIF4A facilitated mainly through binding to the HEAT1 domain of eIF4G. In order to understand the interaction of HEAT1 with eIF4A and other components during translation initiation backbone assignment is essential. Here we report the ¹H, ¹³C and ¹⁵N backbone assignment for the HEAT1 domain of human eIF4G isoform I (eIF4GI-HEAT1), the first of three HEAT domains of eIF4G (29 kDa) as a basis for the elucidation of its structure and interactions with its binding partners, necessary for understanding the mechanism of its biological function.