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Eukaryotic initiation factor (eIF) 4B is part of the protein complex involved in the recognition and binding of mRNA to the ribosome. DrosophilaeIF4B is a single-copy gene that encodes two isoforms, termed eIF4B-L (52.2 kDa) and eIF4B-S (44.2 kDa), generated as a result of the alternative recognition of two polyadeynlation signals during transcription termination and subsequent alternative splicing of the two pre-mRNAs. Both eIF4B mRNAs and proteins are expressed during the entire embryogenesis and life cycle. The proteins are cytoplasmic with polarized distribution. The two isoforms bind RNA with the same affinity. eIF4B-L and eIF4B-S preferentially enhance cap-dependent over IRES-dependent translation initiation in a Drosophila cell-free translation system. RNA interference experiments suggest that eIF4B is required for cell survival, although only a modest reduction in rate of protein synthesis is observed. Overexpression of eIF4B in Drosophila cells in culture and in developing eye imaginal discs promotes cell proliferation.

The ubiquitous intracellular parasite Toxoplasma gondii (phylum Apicomplexa) differentiates into an encysted form (bradyzoite) that can repeatedly re-emerge as a life-threatening acute infection (tachyzoite) upon impairment of immunity. Since the switch from tachyzoite to bradyzoite is a stress-induced response, we sought to identify components related to the phosphorylation of the alpha subunit of eIF2 (eukaryotic initiation factor-2), a well-characterized event associated with stress remediation in other eukaryotic systems. In addition to characterizing Toxoplasma eIF2alpha (TgIF2alpha), we have discovered a novel eIF2 protein kinase, designated TgIF2K-A (Toxoplasma gondii initiation factor-2kinase). Although the catalytic domain of TgIF2K-A contains sequence and structural features that are conserved among members of the eIF2 kinase family, TgIF2K-A has an extended N-terminal region that is highly divergent from other eIF2 kinases. TgIF2K-A specifically phosphorylates the regulatory serine residue of yeast eIF2alpha in vitro and in vivo, and can modulate translation when expressed in the yeast model system. We also demonstrate that TgIF2K-A phosphorylates the analogous regulatory serine residue of recombinant TgIF2alpha in vitro. Finally, we demonstrate that TgIF2alpha phosphorylation in tachyzoites is enhanced in response to heat shock or alkaline stress, conditions known to induce parasite differentiation in vitro. Collectively, this study suggests that eIF2 kinase-mediated stress responses are conserved in Apicomplexa, and a novel family member exists that may control parasite-specific events, including the clinically relevant conversion into bradyzoite cysts.

Eukaryotic initiation factor 4E (eIF4E) has central roles in the control of several aspects of post-transcriptional gene expression and thereby affects developmental processes. It is also implicated in human diseases. This review explores the relationship between structural, biochemical and biophysical aspects of eIF4E and its function in vivo, including both long-established roles in translation and newly emerging ones in nuclear export and mRNA decay pathways.

The translational factor eukaryotic initiation factor 4E (eIF4E) is a central component in the initiation and regulation of translation in eukaryotic cells. Through its interaction with the 5' cap structure of mRNA, eIF4E functions to recruit mRNAs to the ribosome. The accumulation of expressed sequence tag sequences has allowed the identification of three different eIF4E-family members in mammals termed eIF4E-1, eIF4E-2 (4EHP, 4E-LP) and eIF4E-3, which differ in their structural signatures, functional characteristics and expression patterns. Unlike eIF4E-1, which is found in all eukaryotes, orthologues for eIF4E-2 appear to be restricted to metazoans, while those for eIF4E-3 have been found only in chordates. Like prototypical eIF4E-1, eIF4E-2 was found to be ubiquitously expressed, with the highest levels in the testis. Expression of eIF4E-3 was detected only in heart, skeletal muscle, lung and spleen. Similarly to eIF4E-1, both eIF4E-2 and eIF4E-3 can bind to the mRNA cap-structure. However, in contrast to eIF4E-1 which interacts with both the scaffold protein, eIF4G and the translational repressor proteins, the eIF4E-binding proteins (4E-BPs), eIF4E-2 and eIF4E-3 each possesses a range of partial activities. eIF4E-2 does not interact with eIF4G, but does interact with 4E-BPs. Conversely, eIF4E-3 interacts with eIF4G, but not with 4E-BPs. Neither eIF4E-2 nor eIF4E-3 is able to rescue the lethality of eIF4E gene deletion in yeast. It is hypothesized that each eIF4E-family member fills a specialized niche in the recruitment of mRNAs by the ribosome through differences in their abilities to bind cap and/or to interact with eIF4G and the 4E-BPs.

Translation initiation is important for the regulation of both cell growth and cell division. It is uniquely poised to coordinate overall cell proliferation by its effects on both growth and division. A number of translation initiation factors are transcriptional targets of c-myc in a variety of assays. In particular, the mRNA cap-binding protein eIF4E has a myc-binding sequence in its promoter that is myc responsive in reporter assays and contains a high-affinity myc-binding site in chromosome immunoprecipitation experiments. Several differential expression screens have demonstrated altered levels of eIF4E, along with several other translation initiation factors, in response to alterations of c-myc levels. The potential for eIF4E and other translational control elements to mediate myc's transforming functions is particularly important because eIF4E is itself a known oncogenic factor. The ability of translation initiation factors to affect both cell division control and cell growth control coincides with myc's remarkable effects on both cell growth and cell division.

Regulation of protein synthesis is increasingly being recognized as an important determinant of cell proliferation and senescence. In particular, recent evidence indicates that eukaryotic translation initiation factor 5A (eIF-A) plays a pivotal role in this determination. Separate isoforms of eIF-5A appear to facilitate the translation of mRNAs required for cell division and cell death. This raises the possibility that eIF-5A isoforms are elements of a biological switch that is in one position in dividing cells and in another position in dying cells. Changes in the position of this putative switch in response to physiological and environmental cues are likely to have a significant impact on plant growth and development.

Infectious bursal disease virus (IBDV), a member of the family Birnaviridae, is a non-enveloped, double-stranded RNA virus. Viral protein 1 (VP1), the putative RNA-dependent RNA polymerase, occurs in virions both as a free polypeptide and as a genome-linked protein, called VPg. To gain more insight in its function, we initiated a yeast two-hybrid screen. With this approach we identified the carboxy-terminal domain of eukaryotic translation initiation factor 4AII (eIF4AII) as an interactor for VP1. The association between these molecules was confirmed by co-immunoprecipitation analyses. eIF4A plays an essential role in the initiation of translation of both capped and uncapped mRNAs. Its association with IBDV VP1 suggests an involvement of this viral protein in IBDV mRNA translation. An interaction between VP1 and full-length eIF4AII was, however, not observed. In view of the known two-domain structure of eIF4AII it is conceivable that the interaction of VP1 with full-length eIF4AII requires collaborating proteins that open up its structure and expose the VP1-binding site in the carboxy-terminal domain. The biological relevance of the potential VP1-eIF4AII interaction is discussed.

The human GADD34 (Growth Arrest and DNA Damage-inducible 34) is the product of an apoptosis- and DNA-damage-inducible gene. The C-terminus domain of GADD34 is highly homologous to HSV-1 gamma-1 34.5, HSV-2 and the African swine fever virus virulence-associated factor NL-S. Among these viral proteins, HSV-1 gamma 34.5 protein is known to prevent apoptosis of viral-infected cells. Because of the difficulty in expressing GADD34 protein or any of its fragments, including the C-terminus (amino acids 533-632) in E. coli, partially due to sub-optimal expression of eukaryotic codons in prokaryotic E. coli, we used a codon-optimized cloning scheme to construct the eukaryotic gene that codes for GADD34(533-632). We derived a novel PCR protocol to assemble 20 oligonucleotides into the synthetic GADD34(533-632) gene. The clear advantage of using this protocol is that the assembled gene is without the mutation and deletion that are usually of a major problem in constructing synthetic genes. The synthetic GADD34(533-632) gene was cloned, expressed, and purified in large quantity. We obtained approximately 50 mg of GADD34(533-632) protein per liter minimum-medium culture. To our knowledge, this is the first report of a large-scale production of the C-terminus of GADD34. The production and purification of GADD34(533-632) in large quantity are essential for structure determination as well as for understanding its interactions with other proteins such as phosphatase 1-alpha using NMR spectroscopy and other biophysical methods.

The phylogenetically conserved eukaryotic translation initiation factor 5A (eIF5A) is the only known cellular protein to contain the post-translationally derived amino acid hypusine [Ne-(4-amino-2-hydroxybutyl)lysine]. Both eIF5A and its hypusine modification are essential for sustained cell proliferation. Normally only one eIF5A protein is expressed in human cells. Recently, we identified a second human EIF5A gene that would encode an isoform (eIF5A-2) of 84% sequence identity. Overexpression of eIF5A-2 mRNA in certain human cancer cells, in contrast to weak normal expression limited to human testis and brain, suggests EIF5A2 as a potential oncogene. However, eIF5A-2 protein has not been described in human or mammalian cells heretofore. Here, we describe the identification of eIF5A-2 protein in human colorectal and ovarian cancer lines, SW-480 and UACC-1598, that overexpress eIF5A-2 mRNAs. Functional characterization of the human isoforms revealed that either human EIF5A gene can complement growth of a yeast strain in which the yeast EIF5A genes were disrupted. This indicates functional similarity of the human isoforms in yeast and suggests that eIF5A-2 has an important role in eukaryotic cell survival similar to that of the ubiquitous eIF5A-1. Detectable structural differences were also noted, including lack of immunological cross-reactivity, formation of different complexes with deoxyhypusine synthase, and Km values (1.5 ± 0.2 vs. 8.3 ± 1.4 µm for eIF5A-1 and -2, respectively) as substrates for deoxyhypusine synthase in vitro. These physical characteristics and distinct amino acid sequences in the C-terminal domain together with differences in gene expression patterns imply differentiated, tissue-specific functions of the eIF5A-2 isoform in the mammalian organism and in cancer.

Proteins provide the structural framework of a cell and perform the enzymatic activities sustaining DNA replication and energy production. The hormones and growth factors that facilitate organ-to-organ communication are proteins as are the receptors and signaling intermediaries that integrate extracellular stimuli to intracellular action. As such, eukaryotic cells devote tremendous effort and energy to protein synthesis. The enzymes involved in protein synthesis have traditionally been described as cellular housekeepers. This was meant to imply that while they were necessary for cell viability, they were not thought to have a causal role in activating cell differentiation or neoplastic development the way that a transcription factor or hormone receptor might. However, two protein translation factors, protein initiation factor eIF4E and protein elongation factor eEF1A2, have been identified as important human oncogenes. This review summarizes recent work showing that protein initiation and elongation factors have important regulatory roles in cell growth, apoptosis, and tumorigenesis.

Biochemical studies of eukaryotic proteins are often constrained by low availability of these typically large, multicomponent protein complexes in pure form. Escherichia coli is a commonly used host for large-scale protein production; however, its utility for eukaryotic protein production is limited because of problems associated with transcription, translation, and proper folding of proteins. Here we describe the development and testing of pLANT, a vector that addresses many of these problems simultaneously. The pLANT vector contains a T7 promoter-controlled expression unit, a p15A origin of replication, and genes for rare transfer RNAs and kanamycin resistance. Thus, the pLANT vector can be used in combination with the pET vector to coexpress multiple proteins in E. coli. Using this approach, we have successfully produced high-milligram quantities of two different Saccharomyces cerevisiae complexes in E. coli: the heterodimeric Msh2-Msh6 mismatch repair protein (248kDa) and the five-subunit replication factor C clamp loader (250 kDa). Quantitative analyses indicate that these proteins are fully active, affirming the utility of pLANT+pET-based production of eukaryotic proteins in E. coli for in vitro studies of their structure and function.

Amino acids act through a number of signaling pathways and mechanisms to mediate control of gene expression at the level of mRNA translation. This report reviews recent findings that illustrate the manner through which amino acids act to regulate the initiation phase of mRNA translation. The report focuses on signaling pathways that involve the eukaryotic initiation factor-2 (eIF2) protein kinase, general control non-derepressing kinase-2 and the mammalian target of rapamycin (mTOR) protein kinase. It also describes the mechanisms through which amino acid-induced modulation of eIF2 phosphorylation and mTOR-mediated signaling cause derepression of translation of specific mRNAs and result in an overall change in the pattern of gene expression. Finally, it provides examples of mRNAs whose translation is modulated through these mechanisms.

The eukaryotic initiation factor eIF4G is a large modular protein which serves as a docking site for initiation factors and proteins involved in RNA translation. Together with eIF4E and eIF4A, eIF4G constitutes the eIF4F complex which is a key component in promoting ribosome binding to the mRNA. Thus, the central role of eIF4G in initiation makes it a valid target for events aimed at modulating translation. Such events occur during viral infection by picornaviruses and lentiviruses and result in the hijack of the translational machinery through cleavage of eIF4G. Proteolysis of eIF4G is also mediated by caspases during the onset of apoptosis causing inhibition of protein synthesis. We will review the role of eIF4G and protein partners as well as the cellular and viral events that modulate eIF4G activity in the initiation of translation.

The translation initiation factors (IFs) IF1/eIF1A and IF2/eIF5B have been conserved throughout all kingdoms. Although the central roles of the bacterial factors IF1 and IF2 were established long ago, the importance of their eukaryotic homologs, eukaryotic IFs (eIFs) eIF1A and eIF5B, has only recently become evident. The translation machinery in eukaryotes is more complex and accordingly, eIF1A and eIF5B seem to have acquired a number of new functions while also retaining many of the roles of bacterial IF1 and IF2. IF1 and IF2 have been shown to interact on the ribosome but no binding has been detected for the free factors. In contrast, yeast eIF1A and eIF5B have been reported to interact in the absence of ribosomes. Here, we have identified the binding interface between human eIF1A and the C-terminal domain of eIF5B by using solution NMR. That interaction interface involves the C termini of the two proteins, which are not present in bacterial IF1 and IF2. The interaction is, therefore, unique to eukaryotes. A structural model for the interaction of eIF1A and eIF5B in the context of the ribosome is presented. We propose that eIF1A and eIF5B simultaneously interact at two sites that are >50 Å apart: through their C termini as reported here, and through an interface previously identified in bacterial IF1 and IF2. The binding between the C termini of eIF1A and eIF5B has implications for eukaryote-specific mechanisms of recruitment and release of translation IFs from the ribosome.

Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34cdc2-related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis, and apoptotic signaling. However, substrates of CDK11 during apoptosis have not been identified. We used a yeast two-hybrid screening strategy and identified eukaryotic initiation factor 3 p47 protein (eIF3 p47) as an interacting partner of caspase-processed C-terminal kinase domain of CDK11 (CDK11p46). We demonstrate that the eIF3 p47 can interact with CDK11 in vitro and in vivo, and the interaction can be strengthened by stimulation of apoptosis. EIF3 p47 contains a Mov34/JAB domain and appears to interact with CDK11p46 through this motif. We show in vitrothat the caspase-processed CDK11p46 can phosphorylate eIF3 p47 at a specific serine residue (Ser46) and that eIF3 p47 is phosphorylated in vivo during apoptosis. Purified recombinant CDK11p46 inhibited translation of a reporter gene in vitro in a dose-dependent manner. In contrast, a kinase-defective mutant CDK11p46M did not inhibit translation of the reporter gene. Stable expression of CDK11p46 in vivo inhibited the synthesis of a transfected luciferase reporter protein and overall cellular protein synthesis. These data provide insight into the cellular function of CDK11 during apoptosis.

Significant progress has been made over the past several years on structural studies of the eukaryotic translation initiation factors that facilitate the assembly of a translation-competent ribosome at the initiation codon of an mRNA. These structural studies have revealed the repeated use of a set of common structural folds, highlighted the evolutionary conservation of the translation apparatus, and provided insight into the mechanism and regulation of cellular and viral protein synthesis.

The region located downstream of the initiation codon constitutes part of the translation initiation signal, significantly affecting the level of protein expression in E. coli. In order to determine its influence on translation initiation, we inserted random 12-base sequences downstream of the initiation codon of the lacZ gene. A total of 119 random clones showing higher β-galactosidase activities than the control lacZ gene were isolated and subsequently sequenced. Analysis of these clones revealed that their insertion sequences are strikingly rich in A and T, but poor in G, with no consensus sequences among them. Toeprinting experiments and polysome profile analysis confirmed that the A/T-rich sequences enhance translation at the level of initiation. Collectively, the present data demonstrate that A/T richness of the region following the initiation codon plays a significant role in E. coli gene expression.

Initiation factors IF2 in bacteria and eIF2 in eukaryotes are GTPases that bind Met-tRNA \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{i}^{Met}\end{equation*}\end{document} to the small ribosomal subunit. eIF5B, the eukaryotic ortholog of IF2, is a GTPase that promotes ribosomal subunit joining. Here we show that eIF5B GTPase activity is required for protein synthesis. Mutation of the conserved Asp-759 in human eIF5B GTP-binding domain to Asn converts eIF5B to an XTPase and introduces an XTP requirement for subunit joining and translation initiation. Thus, in contrast to bacteria where the single GTPase IF2 is sufficient to catalyze translation initiation, eukaryotic cells require hydrolysis of GTP by both eIF2 and eIF5B to complete translation initiation.

The mammalian GSPT, which consists of amino-terminal (N) and carboxyl-terminal (C) domains, functions as the eukaryotic releasing factor 3 (eRF3) by interacting with eRF1 in translation termination. This function requires only the C-domain that is homologous to the elongation factor (EF) 1alpha, while the N-domain interacts with polyadenylate-binding protein (PABP), which binds the poly(A) tail of mRNA and associates with the eukaryotic initiation factor (eIF) 4G. Here we describe a novel role of GSPT in translation. We first determined an amino acid sequence required for the PABP interaction in the N-domain. Inhibition of this interaction significantly attenuated translation of capped/poly(A)-tailed mRNA not only in an in vitro translation system but also in living cells. There was a PABP-dependent linkage between the termination factor complex eRF1-GSPT and the initiation factor eIF4G associating with 5' cap through eIF4E. Although the inhibition of the GSPT-PABP interaction did not affect the de novo formation of an 80 S ribosomal initiation complex, it appears to suppress the subsequent recycle of ribosome. These results indicate that GSPT/eRF3 plays an important role in translation cycle through the interaction with PABP, in addition to mediating the termination with eRF1.

Hepatitis C virus (HCV) NS5B protein has been shown to have RNA-dependent RNA polymerase (RdRp) activity by itself and is a key enzyme involved in viral replication. Using analyses with the yeast two-hybrid system and in vitro binding assay, we found that human eukaryotic initiation factor 4AII (heIF4AII), which is a component of the eIF4F complex and RNA-dependent ATPase/helicase, interacted with NS5B protein. These two proteins were shown to be partially colocalized in the perinuclear region. The binding site in HCV NS5B protein was localized within amino acid residues 495 to 537 near the C terminus. Since eIF4A has a helicase activity and functions in a bidirectional manner, the binding of HCV NS5B protein to heIF4AII raises the possibility that heIF4AII facilitates the genomic RNA synthesis of NS5B protein by unwinding the secondary structure of the HCV genome and is a host component of viral replication complex.

Protein synthesis is the ultimate step of gene expression and a key control point for regulation. In particular, it enables cells to rapidly manipulate protein production without new mRNA synthesis, processing, or export. Recent studies have enhanced our understanding of the translation initiation process and helped elucidate how modifications of the general translational machinery regulate gene-specific protein production.

The cDNA encoding human heme-regulated eukaryotic initiation factor-2 alpha (eIF-2 alpha) kinase was cloned from a human bone marrow culture. Its deduced amino acid sequence comprised of 629 amino acids with a calculated molecular weight of 71,031 Da. RT-PCR analysis revealed that the gene was also expressed in heart, kidney, spleen, muscle, and stomach.

Aims: We aim to unravel the divergent roles of the paralogs of the ubiquitous mRNA helicase eIF4A ; these crucial proteins have emerged as major therapeutic targets in cancer. Methods: We have used a broad range of epidemiological, tissue-based, cellular and biochemical methods. We have examined expression levels of the paralogs in a large set of lung adenocarcinomas by quantitative multiplex immunofluorescence. Cellular and biochemical effects of eIF4A paralog knockdown have been studied in cell culture using a range of growth and apoptosis assays, alongside polysome profiling, RNA-Seq methods. Reporter assays and structure-probing methods are being used to dissect how the paralogs interact with mRNA structures. Purified proteins are being used in functional assays of helicase/ATPase activity. Introduction: Eukaryotic initiation factor 4A (eIF4A) is a ubiquitous mRNA helicase, encoded by two paralogous genes which are often considered to be functionally redundant mediators of translational initiation, although we now believe that eIF4A2 has distinct roles in the function of micro-RNAs. As eIF4A1 is known to be essential for the translation of many pro-oncogenic mRNAs, eIF4A has emerged as an attractive new cancer target. Results: We find that eIF4A1 and eIF4A2 have extremely divergent cellular roles and clinical associations. High levels of eIF4A1 are found in proliferating/stem cell compartments of normal organs, we find that elevated eIF4A1 predicts poor outcome in lung adenocarcinoma. eIF4A2, in contrast, is expressed in terminally differentiated normal cells and has is related to good outcomes in cancer. Therefore we have been studying the cellular effects of eIF4A1 and eIF4A2 knockdown. eIF4A1 drives cellular proliferation, while eIF4A2 is pro-aoptotic and limits cellular growth. We have identified mRNAs which are influenced by eIF4A1 and eIF4A2 at the levels of mRNA abundance and translational control. eIF4A1 is necessary for the translation of mRNAs with G/C-rich 5' UTRs and the potential for G-quadruplex formation; in contrast eIF4A2 has a generally inhibitory effect upon translation initiation, and influences a different set of mRNAs at both total and translational levels. We find that mRNA elements with the potential for G-quadruplex formation can endow reporter mRNAs with dependence upon eIF4A1 for their translation, but that this is highly dependent upon sequence context, and also upon the size of the predicted quadruplex. Finally, we have generated pure recombinant versions of both paralogs and their cellular binding partners, and are now starting to generate paralog-specific data on their biochemical properties. Conclusions: eIF4A1 and eIF4A2 exhibit great divergence in their pathological, cellular and biochemical roles. eIF4A1 is associated with cancer virulence mediated by upregulation of many pro-proliferative proteins, while eIF4A2 shows the opposite tendency. The sequence and structural determinants of eIF4A-dependence are complex and highly context-dependent. eIF4A1 and eIF4A2 are biochemically distinct in functional assays. These findings are central to our understanding of the control of cellular translation, and have great implications for the therapeutic targeting of translation in cancer. Citation Format: Farheen Raza, Madhumita Das, Joseph Waldron, Kari Kopra, John Le Quesne. eIF4a paralogue switching drives opposing phenotypes in cancer by specific reprogramming of gene expression. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A13.

Eukaryotic translation is a complex process composed of three main steps: initiation, elongation, and termination. During infections by RNA- and DNA-viruses, the eukaryotic translation machinery is used to assure optimal viral protein synthesis. Human immunodeficiency virus type I (HIV-1) uses several non-canonical pathways to translate its own proteins, such as leaky scanning, frameshifting, shunt, and cap-independent mechanisms. Moreover, HIV-1 modulates the host translation machinery by targeting key translation factors and overcomes different cellular obstacles that affect protein translation. In this review, we describe how HIV-1 proteins target several components of the eukaryotic translation machinery, which consequently improves viral translation and replication.

Regulation of protein synthesis is of fundamental importance to cells. It has a critical role in the control of gene expression, and consequently cell growth and proliferation. The importance of this control is supported by the fact that protein synthesis is frequently upregulated in tumor cells. The major point at which regulation occurs is the initiation stage. Initiation of translation involves the interaction of several proteins to form the eIF4F complex, the recognition of the mRNA by this complex, and the subsequent recruitment of the 40S ribosomal subunit to the mRNA. This results in the formation of the 48S complex that then scans the mRNA for the start codon, engages the methionyl-tRNA and eventually forms the mature 80S ribosome which is elongation-competent. Formation of the 48S complex is regulated by the availability of individual initiation factors and through specific protein-protein interactions. Both of these events can be regulated by post-translational modification by ubiquitin or Ubls (ubiquitin-like modifiers) such as SUMO or ISG15. We provide here a summary of translation initiation factors that are modified by ubiquitin or Ubls and, where they have been studied in detail, describe the role of these modifications and their effects on regulating protein synthesis.

Protein synthesis is an essential cellular process that is highly responsive to extra- and intracellular cues such as mitogens, growth signals, nutrient and energy status, and stress. Much regulation of translation is directed towards initiation, more specifically the ribosome recruitment phase which requires the eukaryotic initiation factor (eIF) 4F complex to prepare the mRNA for the incoming 40S ribosome (and associated factors). As many commonly deregulated signaling pathways found in cancers converge to influence protein synthesis, targeting factors involved in translation initiation is a viable anti-neoplastic strategy that has demonstrated success in pre-clinical settings. Furthermore, transcripts that are particularly sensitive to fluctuations in levels of the eIF4F complex are often associated with oncogenic characteristics (eg proliferation, survival, and angiogenesis) and their translational output appears to be preferentially reduced when eIF4F is inhibited. In particular, the enzymatic subunit of the eIF4F complex, eIF4A, has been extensively explored as a druggable target with several natural products identified as potent and selective inhibitors. In this review, we discuss the cellular regulation of eIF4A activity and its potential as a therapeutic target. This article is part of a Special Issue entitled: Translation and Cancer.

Eukaryotic cells rapidly reduce protein synthesis in response to various stress conditions. This can be achieved by the phosphorylation-mediated inactivation of a key translation initiation factor, eukaryotic initiation factor 2 (eIF2). However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response. We carried out ribosome profiling of cultured human cells under conditions of severe stress induced with sodium arsenite. Although this led to a 5.4-fold general translational repression, the protein coding open reading frames (ORFs) of certain individual mRNAs exhibited resistance to the inhibition. Nearly all resistant transcripts possess at least one efficiently translated upstream open reading frame (uORF) that represses translation of the main coding ORF under normal conditions. Site-specific mutagenesis of two identified stress resistant mRNAs (PPP1R15B and IFRD1) demonstrated that a single uORF is sufficient for eIF2-mediated translation control in both cases. Phylogenetic analysis suggests that at least two regulatory uORFs (namely, in SLC35A4 and MIEF1) encode functional protein products. DOI: http://dx.doi.org/10.7554/eLife.03971.001, eLife digest Proteins carry out essential tasks for living cells and genes contain the instructions to make proteins within their DNA. These instructions are copied to make a molecule of mRNA, and a molecular machine known as a ribosome then reads and translates the mRNA to build the protein. The first step in the translation process is called ‘initiation’ and requires a protein called eIF2 to work together with the ribosome. This step involves identifying an instruction called the start codon that marks the beginning of the mRNA's coding sequence. The section of an mRNA molecule before the start codon is not normally translated by the ribosome and is hence called the 5′ untranslated region. Building proteins requires energy and resources, and so it is carefully regulated. If a cell is stressed, such as by being exposed to harmful chemicals, it makes fewer proteins in order to conserve its resources. This down-regulation of protein production is achieved in part by the cell chemically modifying its eIF2 proteins to make them less able to initiate translation. However, stressed cells still continue to make more of certain proteins that help them to combat stress. The mRNA molecules for some of these proteins contain at least one other start codon in the 5′ untranslated region. The sequence that would be translated from such a start codon is known as an upstream open reading frame (or uORF for short)—and this feature is thought to help certain proteins to still be expressed despite low levels of active eIF2. Andreev, O'Connor et al. have now analysed which mRNAs are translated in human cells that have been treated with a chemical that induces stress and makes the eIF2 protein less able to initiate translation. To do so, a technique called ribosome profiling was used to identify all of the mRNA molecules bound to ribosomes shortly after treatment with this chemical. Overall translation of most mRNAs in stressed cells was reduced to a quarter of the normal level. However, Andreev, O'Connor et al. observed that the translation of a few mRNAs continued almost as normal, or even increased, after the chemical treatment. Notably, most of these mRNAs encoded regulatory proteins, which are not required in large amounts. With one exception, all of these resistant mRNAs contained uORFs. In unstressed cells, these uORFs were efficiently translated, while the same mRNA's coding sequences were translated less efficiently. Andreev, O'Connor et al. suggest that these two features could be used to identify mRNAs that are still translated into working proteins when cells are stressed. Further work is now needed to explore the mechanisms by which translation of these uORFs allows mRNAs to resist the stress. DOI: http://dx.doi.org/10.7554/eLife.03971.002

Eukaryotic translation initiation factors are the principal molecular effectors regulating the process converting nucleic acid to functional protein. Commonly referred to as eIFs (eukaryotic initiation factors), this suite of proteins is comprised of at least 25 individual subunits that function in a coordinated, regulated, manner during mRNA translation. Multiple facets of eIF regulation have yet to be elucidated; however, many of the necessary protein factors are phosphorylated. Herein, we have isolated, identified and quantified phosphosites from eIF2, eIF3, and eIF4G generated from log phase grown HeLa cell lysates. Our investigation is the first study to globally quantify eIF phosphosites and illustrates differences in abundance of phosphorylation between the residues of each factor. Thus, identification of those phosphosites that exhibit either high or low levels of phosphorylation under log phase growing conditions may aid researchers to concentrate their investigative efforts to specific phosphosites that potentially harbor important regulatory mechanisms germane to mRNA translation.

Small ubiquitin-like modifier (SUMO) proteins regulate many important eukaryotic cellular processes through reversible covalent conjugation to target proteins. In addition to its many well-known biological consequences, like subcellular translocation of protein, subnuclear structure formation, and modulation of transcriptional activity, we show here that SUMO-2 also plays a role in mRNA translation. SUMO-2 promoted formation of the active eukaryotic initiation factor 4F (eIF4F) complex by enhancing interaction between Eukaryotic Initiation Factor 4E (eIF4E) and Eukaryotic Initiation Factor 4G (eIF4G), and induced translation of a subset of proteins, such as cyclinD1 and c-myc, which essential for cell proliferation and apoptosis. As expected, overexpression of SUMO-2 can partially cancel out the disrupting effect of 4EGI-1, a small molecule inhibitor of eIF4E/eIF4G interaction, on formation of the eIF4F complex, translation of the cap-dependent protein, cell proliferation and apoptosis. On the other hand, SUMO-2 knockdown via shRNA partially impaired cap-dependent translation and cell proliferation and promoted apoptosis. These results collectively suggest that SUMO-2 conjugation plays a crucial regulatory role in protein synthesis. Thus, this report might contribute to the basic understanding of mammalian protein translation and sheds some new light on the role of SUMO in this process.

This chapter summarizes the most important advances concerning structural and mechanistic aspects of translation initiation in bacteria which have occurred since the appearance of the last reviews on this subject. The small ribosomal subunit (30S) interacts with mRNA and fMet-tRNA in stochastic order to yield a bona fide 30S initiation complex through the rearrangement, kinetically controlled by the three initiation factors, of an unstable kinetic intermediate called the pre-ternary complex. Systematic variations of the translation initiation region (TIR) elements in the in vivo translation of reporter genes in E. coli and Bacillus subtilis have produced similar results in two similar analyses. The differences observed in a study between heterologous and homologous proteins and the sometimes contradictory conclusions reached by similar studies concerning the relevance of specific TIR elements and the consequent criteria for optimization of translation remind us once again that each gene (mRNA) might be endowed with particular, not easily predictable properties. The dispensable nature of the Shine-Dalgarno (SD) sequence is evidenced by the existence of leaderless mRNAs which begin directly with an AUG initiation triplet. The recognition and binding of fMet-tRNA by IF2 play crucial roles in the translation initiation pathway of bacteria. The understanding of the translation initiation pathway cannot be considered satisfactory without full elucidation of the role played in this process by GTP and by the IF2-dependent GTPase.

Different from canonical translation initiation, some plant viral RNAs lack a 5’cap (an unusual 7-methyl guanosine linked by a 5’to 5’ triphosphate). They utilize a cap independent translation element (CITE) to efficiently start translation. Barley yellow dwarf virus (BYDV) has a translation element (BTE) located in its 3’UTR mRNA. BTE binds with eukaryotic initiation factor eIF4G (one subunit of heterodimer of eIF4F) and recruits other initiation factors to assemble the ribosome preinitiation complex for synthesis of viral proteins. BYDV is one of the world-wide economically most important viruses causing severe crop loss. How BTE mediate BYDV translation is still unclear. By measuring fluorescence anisotropy of labeled RNA, here we report 3’BTE binds with a truncated fragment of eIF4G, eIF4G601-1196 (contained amino acid residue from 601 to 1196). The equilibrium dissociation constants (Kd) of this binding is 39.5±3.5 nM. Adding eIF4E, the cap binding protein, can increase the binding ability of eIF4G60...

The eukaryotic translation initiation factor 5A (EIF5A) protein is highly conserved in eukaryotes. Some studies shown that eIF5A has important roles in cell proliferation control and HIV-1 replicat...

Gene expression during muscle cell differentiation is tightly regulated at multiple levels, including translation initiation. The PI3K/mTOR signalling pathway exerts control over protein synthesis by regulating assembly of eukaryotic initiation factor (eIF) 4F, a heterotrimeric complex that stimulates recruitment of ribosomes to mRNA templates. One of the subunits of eIF4F, eIF4A, supplies essential helicase function during this phase of translation. The presence of two cellular eIF4A isoforms, eIF4AI and eIF4AII, has long thought to impart equivalent functions to eIF4F. However, recent experiments have alluded to distinct activities between them. Herein, we characterize distinct regulatory mechanisms between the eIF4A isoforms during muscle cell differentiation. We find that eIF4AI levels decrease during differentiation whereas eIF4AII levels increase during myofiber formation in a MyoD-dependent manner. This study characterizes a previously undefined mechanism for eIF4AII regulation in differentiation and highlights functional differences between eIF4AI and eIF4AII. Finally, RNAi-mediated alterations in eIF4AI and eIF4AII levels indicate that the myogenic process can tolerate short term reductions in eIF4AI or eIF4AII levels, but not both.

The Int-6 protein has been shown to be a subunit of eukaryotic translation initiation factor 3 (eIF3) and to play a role in the control of cell growth. By immunoprecipitation experiments and mass spectrometry analyses, we identified a human protein previously known as HSPC021 that is associated with Int-6. Exposure of Jurkat cells to the phosphatase inhibitor H(2)O(2) triggers a marked phosphorylation on tyrosine of HSPC021. Several experiments were performed to evaluate whether this protein is associated with eIF3. It was observed that HSPC021 coelutes with Int-6 and eIF3 in gel filtration, coimmunoprecipitates with eIF3, and is incorporated into eIF3 both in rabbit reticulocyte lysates and in COS7 cells. A direct protein-protein interaction occurs between HSPC021 and Int-6, but the analysis of different mutants of HSPC021 indicated that a larger region of the protein is necessary for incorporation into eIF3 as compared with binding to Int-6. Taken together, our results establish that HSPC021 is tightly associated with the mammalian translation initiation factor eIF3. Analysis of the primary sequence of HSPC021 from different species revealed the presence of a tetratricopeptide repeat, a proteasome-COP9 (constitutive photomorphogenesis 9) signalosome-initiation factor 3 domain along with a Pumilio FBF repeat. These protein motifs are also present in subunits of eIF3, of the lid of the 26 S proteasome, and of the COP9 signalosome.

Several animal viruses inhibit host protein synthesis, but only some members of the picornavirus group are known to do so by cleaving translation initiation factor eIF4G. Here we report that infection of human CD4 + cells with HIV-1 also leads to proteolysis of eIF4G and profound inhibition of cellular translation. Purified HIV-1 protease directly cleaves eIF4GI at positions 678, 681, and 1086, separating the three domains of this initiation factor. Proteolysis of eIF4GI by HIV-1 protease, as with poliovirus 2A protease, inhibits protein synthesis directed by capped mRNAs but allows internal ribosome entry site-driven translation. These findings indicate that HIV-1, a member of retrovirus group, shares with picornaviruses the capacity to proteolyze eIF4G.

Recent evidence from our laboratory demonstrates that platelets synthesize numerous proteins in a signal-dependent fashion (Pabla, R., Weyrich, A. S., Dixon, D. A., Bray, P. F., McIntyre, T. M., Prescott, S. M., and Zimmerman, G. A. (1999) J. Cell Biol. 144, 175–184; Weyrich, A. S., Dixon, D. A., Pabla, R., Elstad, M. R., McIntyre, T. M., Prescott, S. M., and Zimmerman, G. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5556–5561). Protein synthesis in platelets is controlled at the translational level; however, the mechanisms of regulation are not known. Here we demonstrate that translation initiation factors are redistributed to mRNA-rich areas in aggregated platelets, an event that induces protein synthesis. Interrogation of cDNA arrays revealed that platelet-derived mRNAs are primarily associated with the cytoskeletal core. In contrast, eukaryotic initiation factor 4E (eIF4E), the essential mRNA cap-binding protein that controls global translation rates, is localized in the membrane skeleton and soluble fraction of platelets, physically separated from most mRNAs. Platelet activation redistributes eIF4E to the cytoskeleton and increases interactions of eIF4E with mRNA cap structures. Redistribution of eIF4E to the mRNA-rich cytoskeleton coincides with a marked increase in protein synthesis, a process that is blocked when intracellular actin is disrupted. Additional studies demonstrated that β3integrins are the primary membrane receptor that distributes eIF4E within the cell. These results imply that integrins link receptor-mediated pathways with mRNA-rich cytoskeletal domains and thereby modulate the organization of intracellular translational complexes. They also indicate that the functional status of eIF4E is regulated by its intracellular distribution.

Three protein complexes (the proteasome regulatory lid, the COP9 signalosome and eukaryotic translation initiation factor 3) contain protein subunits with a well defined protein domain, the PCI domain. At least two (the COP9 signalosome and the lid) appear to share a common evolutionary origin. Recent advances in our understanding of the structure and function of the three complexes point to intriguing and unanticipated connections between the cellular functions performed by these three protein assemblies, especially between translation initiation and proteolytic protein degradation.

Gene expression is controlled by interactions between activators and coactivators. These interactions in turn are regulated by signaling pathways and by chromatin remodeling events. Recent studies indicate that the final arbiter of gene regulation is a coactivator scaffold at the promoter.

Formation of the initiation translation complex containing the three initiation factors, IF1, IF2, and IF3, tRNA(fMet), and GTP constitutes the earliest event in the protein synthesis. IF2, a GTP-binding protein, is the principal factor involved in selecting and binding fMet-tRNA(fMet) to the 30 S ribosomal subunit. Although some chloroplast initiation translational factors have been identified and purified from algae, none of these factors have been characterized from plants. In this work, we report the molecular characterization of a nuclear-encoded chloroplastic IF2 gene from common bean (PvIF2cp). We show that the PvIF2cp gene encodes a protein containing a chloroplast translocation signal peptide, able to target a green fluorescent protein fusion protein to chloroplasts. A high accumulation of PvIF2cp transcript was found in photosynthetic tissues, whereas low mRNA levels were detected in etiolated plants and in nonphotosynthetic organs. Additional data indicate that the PvIF2cp transcript accumulation is modulated by light. The PvIF2cp gene encodes a functional factor, since the PvIF2cp conserved region, containing the G-domain and the C-terminal end, complements an Escherichia coli infB null mutation. Phylogenetic analysis using the PvIF2cp conserved region suggests that the PvIF2cp gene originated via endosymbiotic gene transfer to the nucleus and that it may be a useful marker for phylogeny reconstruction.

IF3 is essential for ensuring the fidelity of the initiation step of translation in bacterial cells. Mutations at residues R99 and R131 in the C-terminal domain of the factor have previously been shown to increase initiation from the non-canonical GUA codon. Here we show that these mutant forms of IF3 fail to discriminate against initiation from many different non-AUG codons. They also enhance the activity of mutant tRNAs carrying changes in the three consecutive G-C pairs that are conserved in the anticodon stem of initiator tRNAs. In addition, the IF3 mutants stimulate initiations from leaderless mRNAs and from internal initiation codons, in the absence of any SD-anti-SD interaction. These results indicate that IF3 ensures the accuracy of initiation by inspecting both the codon-anticodon pairing and unique features of the initiator tRNA as well as suppressing initiation from other potential start sites within the mRNA.

We have identified a gene encoding a eukaryotic initiation factor 4E-binding protein (4E-BP) in the EST database of the Dictyostelium cDNA project. The Dictyostelium 4E-BP, designated febA ( f our e - b inding), showed significant similarity to mammalian 4E-BPs. Northern blot analysis revealed that febA was expressed at a high level in the vegetative growth phase but the level of expression decreased during late development. The gene was shown to be non-essential since disruption of the gene had no severe effect; the null mutant proliferated normally and formed normal fruiting bodies. However, strains overexpressing the gene could not be established, suggesting that an excess of FebA protein may have a lethal effect on the cells.

International audience; Post-transcriptional and post-translational modifications of factors involved in translation are very important for the control and accuracy of protein biosynthesis. Among these factors, tRNAs harbor the largest variety of grafted chemical structures, which participate in tRNA stability or mRNA decoding. Here, we focused on Trm112 protein, which associates with four different eukaryotic methyltrans-ferases modifying tRNAs (Trm9 and Trm11) but also 18S-rRNA (Bud23) and translation termination factor eRF1 (Mtq2). In particular, we have investigated the role of Trm112 in the Trm11-Trm112 complex, which forms 2-methylguanosine at position 10 on several tRNAs and thereby is assumed to stabilize tRNA structure. We show that Trm112 is important for Trm11 enzymatic activity by influencing S-adenosyl-L-methionine binding and by contributing to tRNA binding. Using hydrogen-deuterium eXchange coupled to mass spectrometry, we obtained experimental evidences that the Trm11-Trm112 interaction relies on the same molecular bases as those described for other Trm112-methyltransferases complexes. Hence, all Trm112-dependent methyltrans-ferases compete to interact with this partner.

A full length cDNA for the beta subunit of chick (Gallus gallus) eukaryotic translation initiation factor-2 is described. This cDNA was isolated by screening a chick cDNA library with a probe derived via differential display of developing chick heart tissue. Up-regulated expression of eIF-2 beta mRNA was confirmed by reverse Northern dot blot analysis. eIF-2 beta, together with eIF-2 alpha and eIF-2 gamma, comprise subunits of a complex that promotes the binding of methionyl-tRNA to ribosomes during the initiation of protein translation. The nucleotide sequence of the chick eIF-2 beta cDNA predicts a protein of 334 amino acids that has 95%, 93%, 56% and 37% sequence identity with rabbit, human, drosophila and yeast eIF-2 beta, respectively. The deduced eIF-2 beta protein contains a number of functional motifs and domains consistent with the putative function of this protein; these include a potential C2-C2 zinc-finger binding domain, three polylysine regions, and three acidic regions.

Modern biological scientists employ numerous approaches for solving their problems. Among these approaches, the gene fusion is surely one of the well-established valuable tools in various fields of biological sciences. A wide range of applications have been developed to analyze a variety of biological phenomena such as transcriptional regulation, pre-mRNA processing, mRNA decay, translation, protein localization and even protein transport in both prokaryotic and eukaryotic organisms. Gene fusions were also used for the study of protein purification, protein structure, protein folding, protein-protein interaction and protein-DNA interaction. Here, we describe applications of gene fusion technology using the Saccharomyces cerevisiae PHO5 gene encoding repressible acid phosphatase to molecular genetics and biotechnology in S. cerevisiae. Using the PHO5 gene fusion as a reporter, we have identified several cis- and trans-acting genes of S. cerevisiae which are involved in splicing of pre-mRNA, biosynthesis of amino acids, ubiquitin-dependent protein degradation, signal transduction of oxygen and unsaturated fatty acid, regulation of transcription by the nucleosome and chromatin. The PHO5 gene fusions exhibiting the mating-type specific expression were also generated to develop a breeding technique for industrial yeast. It is concluded that the PHO5 gene fusion is extremely useful and should be further exploited to investigate various cellular steps of the eukaryotic gene expression.

Eukaryotic translation initiation factor 5A (eIF5A) is an essential protein tightly linked to cellular polyamine homeostasis. It receives the unique spermidine-derived posttranslational modification hypusine that is necessary for eIF5A's biochemical activity and cellular proliferation. The eIF5A protein stimulates ribosomal peptidyl-transferase and may be involved in nucleocytoplasmic mRNA transport. Little is known about the molecular genetics of eIF5A. Here we report on the sequence and molecular characterization of human EIF5A2, a novel phylogenetically conserved gene for eIF5A. EIF5A2 stretches over 17 kb and consists of five exons and four introns. It is localized at 3q25-q27, often noted for chromosomal instability in cancers. EIF5A2 is highly expressed in testis and colorectal adenocarcinoma and at moderate levels in the brain, in contrast to the ubiquitously expressed EIF5A1 gene. Two EIF5A2 mRNAs share a 129-nt 5' UTR and a coding sequence for the 153-amino-acid eIF5AII protein, but possess two alternative 3' UTRs of 46 and 890 nt that arise through differential polyadenylation. The protein is 84% identical and 94% similar to eIF5AI. Both EIF5A genes are conserved in vertebrates. Our findings lend further support for a specialized gene expression program of polyamine metabolic proteins and regulators that function to maintain polyamine homeostasis at elevated levels during spermatogenesis.

Synonymous codons encode the same amino acid, but differ in other biophysical properties. The evolutionary selection of codons whose properties are optimal for a cell generates the phenomenon of codon bias. Although recent studies have shown strong effects of codon usage changes on protein expression levels and cellular physiology, no translational control mechanism is known that links codon usage to protein expression levels. Here, we demonstrate a novel translational control mechanism that responds to the speed of ribosome movement immediately after the start codon. High initiation rates are only possible if start codons are liberated sufficiently fast, thus accounting for the observation that fast codons are overrepresented in highly expressed proteins. In contrast, slow codons lead to slow liberation of the start codon by initiating ribosomes, thereby interfering with efficient translation initiation. Codon usage thus evolved as a means to optimise translation on individual mRNAs, as well as global optimisation of ribosome availability.