Engineered transient and stable overexpression of translation factors eIF3i and eIF3c in CHOK1 and HEK293 cells gives enhanced cell growth associated with increased c-Myc expression and increased recombinant protein synthesis.

There is a desire to engineer mammalian host cell lines to improve cell growth/biomass accumulation and recombinant biopharmaceutical protein production in industrially relevant cell lines such as the CHOK1 and HEK293 cell lines. The over-expression of individual subunits of the eukaryotic translation factor eIF3 in mammalian cells has previously been shown to result in oncogenic properties being imparted on cells, including increased cell proliferation and growth and enhanced global protein synthesis rates. Here we report on the engineering of CHOK1 and HEK cells to over-express the eIF3i and eIF3c subunits of the eIF3 complex and the resultant impact on cell growth and a reporter of exogenous recombinant protein production. Transient over-expression of eIF3i in HEK293 and CHOK1 cells resulted in a modest increase in total eIF3i amounts (maximum 40% increase above control) and an approximate 10% increase in global protein synthesis rates in CHOK1 cells. Stable over-expression of eIF3i in CHOK1 cells was not achievable, most likely due to the already high levels of eIF3i in CHO cells compared to HEK293 cells, but was achieved in HEK293 cells. HEK293 cells engineered to over-express eIF3i had faster growth that was associated with increased c-Myc expression, achieved higher cell biomass and gave enhanced yields of a reporter of recombinant protein production. Whilst CHOK1 cells could not be engineered to over-express eIF3i directly, they could be engineered to over-express eIF3c, which resulted in a subsequent increase in eIF3i amounts and c-Myc expression. The CHOK1 eIF3c engineered cells grew to higher cell numbers and had enhanced cap- and IRES-dependent recombinant protein synthesis. Collectively these data show that engineering of subunits of the eIF3 complex can enhance cell growth and recombinant protein synthesis in mammalian cells in a cell specific manner that has implications for the engineering or selection of fast growing or high producing cells for production of recombinant proteins.

Protein Synthesis and Translational Control: A Historical Perspective.

Protein synthesis and its regulation are central to all known forms of life and impinge on biological arenas as varied as agriculture, biotechnology, and medicine. Otherwise known as translation and translational control, these processes have been investigated with increasing intensity since the middle of the 20th century, and in increasing depth with advances in molecular and cell biology. We review the origins of the field, focusing on the underlying concepts and early studies of the cellular machinery and mechanisms involved. We highlight key discoveries and events on a timeline, consider areas where current research has engendered new ideas, and conclude with some speculation on future directions for the field.

Principles of Translational Control.

Protein synthesis involves a complex machinery comprising numerous proteins and RNAs joined by noncovalent interactions. Its function is to link long chains of amino acids into proteins with precise sequences as encoded by the genome. Regulation of protein synthesis, called translational control, occurs both at a global level and at specific messenger RNAs (mRNAs). To understand how translation is regulated, knowledge of the molecular structures and kinetic interactions of its components is needed. This review focuses on the targets of translational control and the mechanisms employed.

Mapping surface residues of eIF5A that are important for binding to the ribosome using alanine scanning mutagenesis.

The translation elongation factor eIF5A is conserved through evolution and is necessary to rescue the ribosome during translation elongation of polyproline-containing proteins. Although the site of eIF5A binding to the ribosome is known, no systematic analysis has been performed so far to determine the important residues on the surface of eIF5A required for ribosome binding. In this study, we used clustered charged-to-alanine mutagenesis and structural modeling to address this question. We generated four new mutants of yeast eIF5A: tif51A-4, tif51A-6, tif51A-7 and tif51A-11, and complementation analysis revealed that tif51A-4 and tif51A-7 could not sustain cell growth in a strain lacking wild-type eIF5A. Moreover, the allele tif51A-4 also displayed negative dominance over wild-type eIF5A. Both in vivo GST-pulldowns and in vitro fluorescence anisotropy demonstrated that eIF5A from mutant tif51A-7 exhibited an importantly reduced affinity for the ribosome, implicating the charged residues in cluster 7 as determinant features on the eIF5A surface for contacting the ribosome. Notably, modified eIF5A from mutant tif51A-4, despite exhibiting the most severe growth phenotype, did not abolish ribosome interactions as with mutant tif51A-7. Taking into account the modeling eIF5A + 80S + P-tRNA complex, our data suggest that interactions of eIF5A with ribosomal protein L1 are more important to stabilize the interaction with the ribosome as a whole than the contacts with P-tRNA. Finally, the ability of eIF5A from tif51A-4 to bind to the ribosome while potentially blocking physical interaction with P-tRNA could explain its dominant negative phenotype.

Evidence for a Negative Cooperativity between eIF5A and eEF2 on Binding to the Ribosome.

eIF5A is the only protein known to contain the essential and unique amino acid residue hypusine. eIF5A functions in both translation initiation due to its stimulation of methionyl-puromycin synthesis and translation elongation, being highly required for peptide-bound formation of specific ribosome stalling sequences such as poly-proline. The functional interaction between eIF5A, tRNA, and eEF2 on the surface of the ribosome is further clarified herein. Fluorescence anisotropy assays were performed to determine the affinity of eIF5A to different ribosomal complexes and reveal its interaction exclusively and directly with the 60S ribosomal subunit in a hypusine-dependent manner (Ki60S-eIF5A-Hyp = 16 nM, Ki60S-eIF5A-Lys = 385 nM). A 3-fold increase in eIF5A affinity to the 80S is observed upon charged-tRNAiMet binding, indicating positive cooperativity between P-site tRNA binding and eIF5A binding to the ribosome. Previously identified conditional mutants of yeast eIF5A, eIF5AQ22H/L93F and eIF5AK56A, display a significant decrease in ribosome binding affinity. Binding affinity between ribosome and eIF5A-wild type or mutants eIF5AK56A, but not eIF5AQ22H/L93F, is impaired in the presence of eEF2 by 4-fold, consistent with negative cooperativity between eEF2 and eIF5A binding to the ribosome. Interestingly, high-copy eEF2 is toxic only to eIF5AQ22H/L93F and causes translation elongation defects in this mutant. These results suggest that binding of eEF2 to the ribosome alters its conformation, resulting in a weakened affinity of eIF5A and impairment of this interplay compromises cell growth due to translation elongation defects.

The translation factor eIF5A and human cancer.

The eukaryotic initiation factor eIF5A is a translation factor that, unusually, has been assigned functions in both initiation and elongation. Additionally, it is implicated in transcription, mRNA turnover and nucleocytoplasmic transport. Two eIF5A isoforms are generated from distinct but related genes. The major isoform, eIF5A1, is considered constitutive, is abundantly expressed in most cells, and is essential for cell proliferation. The second isoform, eIF5A2, is expressed in few normal tissues but is highly expressed in many cancers and has been designated a candidate oncogene. Elevated expression of either isoform carries unfavorable prognostic implications for several cancers, and both have been advanced as cancer biomarkers. The amino acid hypusine, a presumptively unique eIF5A post-translational modification, is required for most known eIF5A functions and it renders eIF5A susceptible to inhibitors of the modification pathway as therapeutic targets. eIF5A has been shown to regulate a number of gene products specifically, termed the eIF5A regulon, and its role in translating proline-rich sequences has recently been identified. A model is advanced that accommodates eIF5A in both the initiation and elongation phases of translation. We review here the biochemical functions of eIF5A, the relationship of its isoforms with human cancer, and evolving clinical applications. This article is part of a Special Issue entitled: Translation and Cancer.

The role of eIF3 and its individual subunits in cancer.

Specific individual subunits of eIF3 are elevated or reduced in numerous human tumors, and their ectopic overexpression in immortal cells can result in malignant transformation. The structure and assembly of eIF3 and its role in promoting mRNA and methionyl-tRNAi binding to the ribosome during the initiation phase of protein synthesis are described. Methods employed to detect altered levels of eIF3 subunits in cancers are critically evaluated in order to conclude rigorously that such subunits may cause malignant transformation. Strong evidence is presented that the individual overexpression of eIF3 subunits 3a, 3b, 3c, 3h, 3i and 3m may cause malignant transformation, whereas underexpression of subunits 3e and 3f may cause a similar outcome. Possible mechanisms to explain the malignant phenotypes are examined. The involvement of eIF3 in cancer reinforces the view that translational control plays an important role in the regulation of cell proliferation, and provides new targets for the development of therapeutic agents. This article is part of a Special Issue entitled: Translation and Cancer.

The chaperonin CCT interacts with and mediates the correct folding and activity of three subunits of translation initiation factor eIF3: b, i and h.

eIF3 (eukaryotic initiation factor 3) is the largest and most complex eukaryotic mRNA translation factor in terms of the number of protein components or subunits. In mammals, eIF3 is composed of 13 different polypeptide subunits, of which five, i.e. a, b, c, g and i, are conserved and essential in vivo from yeasts to mammals. In the present study, we show that the eukaryotic cytosolic chaperonin CCT [chaperonin containing TCP-1 (tailless complex polypeptide 1)] binds to newly synthesized eIF3b and promotes the correct folding of eIF3h and eIF3i. Interestingly, overexpression of these last two subunits is associated with enhanced translation of specific mRNAs over and above the general enhancement of global translation. In agreement with this, our data show that, as CCT is required for the correct folding of eIF3h and eIF3i subunits, it indirectly influences gene expression with eIF3i overexpression enhancing both cap- and IRES (internal ribosome entry segment)-dependent translation initiation, whereas eIF3h overexpression selectively increases IRES-dependent translation initiation. Importantly, these studies demonstrate the requirement of the chaperonin machinery for the correct folding of essential components of the translational machinery and provide further evidence of the close interplay between the cell environment, cell signalling, cell proliferation, the chaperone machinery and translational apparatus.

Human eukaryotic initiation factor 4G (eIF4G) protein binds to eIF3c, -d, and -e to promote mRNA recruitment to the ribosome.

Recruitment of mRNA to the 40S ribosomal subunit requires the coordinated interaction of a large number of translation initiation factors. In mammals, the direct interaction between eukaryotic initiation factor 4G (eIF4G) and eIF3 is thought to act as the molecular bridge between the mRNA cap-binding complex and the 40S subunit. A discrete ∼90 amino acid domain in eIF4G is responsible for binding to eIF3, but the identity of the eIF3 subunit(s) involved is less clear. The eIF3e subunit has been shown to directly bind eIF4G, but the potential role of other eIF3 subunits in stabilizing this interaction has not been investigated. It is also not clear if the eIF4A helicase plays a role in stabilizing the interaction between eIF4G and eIF3. Here, we have used a fluorescence anisotropy assay to demonstrate that eIF4G binds to eIF3 independently of eIF4A binding to the middle region of eIF4G. By using a site-specific cross-linking approach, we unexpectedly show that the eIF4G-binding surface in eIF3 is comprised of the -c, -d and -e subunits. Screening multiple cross-linker positions reveals that eIF4G contains two distinct eIF3-binding subdomains within the previously identified eIF3-binding domain. Finally, by employing an eIF4G-dependent translation assay, we establish that both of these subdomains are required for efficient mRNA recruitment to the ribosome and stimulate translation. Our study reveals unexpected complexity to the eIF3-eIF4G interaction that provides new insight into the regulation of mRNA recruitment to the human ribosome.

Introduction to Translation.

We introduce here the inaugural issue of the new scientific journal Translation. The overarching aim of this endeavor is to establish a new forum for a broad spectrum of research in the area of protein synthesis in living systems ranging from structural biochemical, evolutionary and regulatory aspects of translation to the fundamental questions related to post-translational control of somatic phenomena in multicellular organisms including human behavior and health. The journal will publish high quality research articles, provide novel insights, ask provocative questions and discuss new hypothesis in this emerging field. Launching a new journal is always challenging. We hope that strong criteria for the peer-review process, transparency of the editorial policy and the scientific reputation of its founders, editors and editorial board assure the success of Translation; and we rely on continuing support of the scientific community in all aspects of the journal's activity.

Principles of translational control: an overview.

Translational control plays an essential role in the regulation of gene expression. It is especially important in defining the proteome, maintaining homeostasis, and controlling cell proliferation, growth, and development. Numerous disease states result from aberrant regulation of protein synthesis, so understanding the molecular basis and mechanisms of translational control is critical. Here we outline the pathway of protein synthesis, with special emphasis on the initiation phase, and identify areas needing further clarification. Features of translational control are described together with numerous specific examples, and we discuss prospects for future conceptual advances.

The human translation initiation multi-factor complex promotes methionyl-tRNAi binding to the 40S ribosomal subunit.

The delivery of Met-tRNA(i) to the 40S ribosomal subunit is thought to occur by way of a ternary complex (TC) comprising eIF2, GTP and Met-tRNA(i). We have generated from purified human proteins a stable multifactor complex (MFC) comprising eIF1, eIF2, eIF3 and eIF5, similar to the MFC reported in yeast and plants. A human MFC free of the ribosome also is detected in HeLa cells and rabbit reticulocytes, indicating that it exists in vivo. In vitro, the MFC-GTP binds Met-tRNA(i) and delivers the tRNA to the ribosome at the same rate as the TC. However, MFC-GDP shows a greatly reduced affinity to Met-tRNA(i) compared to that for eIF2-GDP, suggesting that MFC components may play a role in the release of eIF2-GDP from the ribosome following AUG recognition. Since an MFC-Met-tRNA(i) complex is detected in cell lysates, it may be responsible for Met-tRNA(i)-40S ribosome binding in vivo, possibly together with the TC. However, the MFC protein components also bind individually to 40S ribosomes, creating the possibility that Met-tRNA(i) might bind directly to such 40S-factor complexes. Thus, three distinct pathways for Met-tRNA(i) delivery to the 40S ribosomal subunit are identified, but which one predominates in vivo remains to be elucidated.

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, eIF2ß, 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 ß-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.

Eukaryotic translation initiation factor (eIF) 5A stimulates protein synthesis in Saccharomyces cerevisiae.

Within the field of eukaryotic protein synthesis, one factor remained putative for decades: eukaryotic translation initiation factor (eIF) 5A. Because eIF5A is an essential protein required for cell proliferation, and one easily targeted by inhibitors, identifying its role in the cell remains important and urgent. Recent reports support early findings that eIF5A stimulates protein synthesis and newly assign the factor a role in elongation rather than initiation. Here we show that eIF5A directly stimulates protein synthesis on native mRNAs, that rapid depletion of eIF5A in vivo immediately leads to a 2-fold inhibition of protein synthesis, and that both the immediate and lasting effects of eIF5A depletion are a reduction in polysome size concomitant with eIF5A depletion. Addition of purified eIF5A to a depleted lysate results in a roughly 2-fold stimulation of protein synthesis in vitro, a result consistent with both older methionyl-puromycin synthesis data and more recently published findings. We find that although eIF5A is not required for protein synthesis, it stimulates the process by about 2- to 3-fold. Our data, along with other published results, reinforce the conclusion that eIF5A stimulates protein synthesis with one important difference: Polysome profiles observed immediately after eIF5A depletion are diagnostic for a role in initiation. This discrepancy is discussed.

Initiation of translation of the FMR1 mRNA Occurs predominantly through 5'-end-dependent ribosomal scanning.

The fragile X mental retardation 1 (FMR1) gene contains a CGG repeat within its 5' untranslated region (5'UTR) that, when expanded to 55-200 CGG repeats (premutation allele), can result in the late-onset neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome. The CGG repeat is expected to form a highly stable secondary structure that is capable of inhibiting 5'-cap-dependent translation. Paradoxically, translation in vivo is only mildly impaired within the premutation range, suggesting that other modes of translation initiation may be operating. To address this issue, we translated in vitro a set of reporter mRNAs containing between 0 and 99 CGG repeats in either native (FMR1) or unrelated (heterologous) 5'UTR context. The 5'-cap dependence of translation was assessed by inserting a stable hairpin (HP) near the 5' end of the mRNAs. The results of the current studies indicate that translation initiation of the FMR1 mRNA occurs primarily by scanning, with little evidence of internal ribosome entry or shunting. Additionally, the efficiency of translation initiation depends on transcription start site selection, with the shorter 5'UTR (downstream transcription start site I) translating with greater efficiency compared to the longer mRNA (start site III) for all CGG-repeat elements studied. Lastly, an HP previously shown to block translation gave differing results depending on the 5'UTR context, in one case initiating translation from within the HP.

Regulation of protein synthesis and the role of eIF3 in cancer.

Maintenance of cell homeostasis and regulation of cell proliferation depend importantly on regulating the process of protein synthesis. Many disease states arise when disregulation of protein synthesis occurs. This review focuses on mechanisms of translational control and how disregulation results in cell malignancy. Most translational controls occur during the initiation phase of protein synthesis, with the initiation factors being the major target of regulation through their phosphorylation. In particular, the recruitment of mRNAs through the m7G-cap structure and the binding of the initiator methionyl-tRNA(i) are frequent targets. However, translation, especially of specific mRNAs, may also be regulated by sequestration into processing bodies or stress granules, by trans-acting proteins or by microRNAs. When the process of protein synthesis is hyper-activated, weak mRNAs are translated relatively more efficiently, leading to an imbalance of cellular proteins that promotes cell proliferation and malignant transformation. This occurs, for example, when the cap-binding protein, eIF4E, is overexpressed, or when the methionyl-tRNA(i)-binding factor, eIF2, is too active. In addition, enhanced activity of eIF3 contributes to oncogenesis. The importance of the translation initiation factors as regulators of protein synthesis and cell proliferation makes them potential therapeutic targets for the treatment of cancer.

Regulation of protein synthesis and the role of eIF3 in cancer

Maintenance of cell homeostasis and regulation of cell proliferation depend importantly on regulating the process of protein synthesis. Many disease states arise when disregulation of protein synthesis occurs. This review focuses on mechanisms of translational control and how disregulation results in cell malignancy. Most translational controls occur during the initiation phase of protein synthesis, with the initiation factors being the major target of regulation through their phosphorylation. In particular, the recruitment of mRNAs through the m7G-cap structure and the binding of the initiator methionyl-tRNAi are frequent targets. However, translation, especially of specific mRNAs, may also be regulated by sequestration into processing bodies or stress granules, by trans-acting proteins or by microRNAs. When the process of protein synthesis is hyper-activated, weak mRNAs are translated relatively more efficiently, leading to an imbalance of cellular proteins that promotes cell proliferation and malignant transformation. This occurs, for example, when the cap-binding protein, eIF4E, is overexpressed, or when the methionyl-tRNAi-binding factor, eIF2, is too active. In addition, enhanced activity of eIF3 contributes to oncogenesis. The importance of the translation initiation factors as regulators of protein synthesis and cell proliferation makes them potential therapeutic targets for the treatment of cancer.

Translation of the FMR1 mRNA is not influenced by AGG interruptions.

The fragile X mental retardation 1 (FMR1) gene contains a CGG-repeat element within its 5' untranslated region (5'UTR) which, for alleles with more than approximately 40 repeats, increasingly affects both transcription (up-regulation) and translation (inhibition) of the repeat-containing RNA with increasing CGG-repeat length. Translational inhibition is thought to be due to impaired ribosomal scanning through the CGG-repeat region, which is postulated to form highly stable secondary/tertiary structure. One striking difference between alleles in the premutation range (55-200 CGG repeats) and those in the normal range (< approximately 40 repeats) is the reduced number/absence of 'expansion stabilizing' AGG interruptions in the larger alleles. Such interruptions, which generally occur every 9-11 repeats in normal alleles, are thought to disrupt the extended CGG-repeat hairpin structure, thus facilitating translational initiation. To test this hypothesis, we have measured the translational efficiency of CGG-repeat mRNAs with 0-2 AGG interruptions, both in vitro (rabbit reticulocyte lysates) and in cell culture (HEK-293 cells). We demonstrate that the AGG interruptions have no detectable influence on translational efficiency in either a cell-free system or cell culture, indicating that any AGG-repeat-induced alterations in secondary/tertiary structure, if present, do not involve the rate-limiting step(s) in translational initiation.