Human eukaryotic initiation factor 4E (eIF4E) and the nucleotide-bound state of eIF4A regulate eIF4F binding to RNA.

During translation initiation, the underlying mechanism by which the eukaryotic initiation factor (eIF) 4E, eIF4A, and eIF4G components of eIF4F coordinate their binding activities to regulate eIF4F binding to mRNA is poorly defined. Here, we used fluorescence anisotropy to generate thermodynamic and kinetic frameworks for the interaction of uncapped RNA with human eIF4F. We demonstrate that eIF4E binding to an autoinhibitory domain in eIF4G generates a high-affinity binding conformation of the eIF4F complex for RNA. In addition, we show that the nucleotide-bound state of the eIF4A component further regulates uncapped RNA binding by eIF4F, with a four-fold decrease in the equilibrium dissociation constant observed in the presence versus the absence of ATP. Monitoring uncapped RNA dissociation in real time reveals that ATP reduces the dissociation rate constant of RNA for eIF4F by ∼4-orders of magnitude. Thus, release of ATP from eIF4A places eIF4F in a dynamic state that has very fast association and dissociation rates from RNA. Monitoring the kinetic framework for eIF4A binding to eIF4G revealed two different rate constants that likely reflect two conformational states of the eIF4F complex. Furthermore, we determined that the eIF4G autoinhibitory domain promotes a more stable, less dynamic, eIF4A-binding state, which is overcome by eIF4E binding. Overall, our data support a model whereby eIF4E binding to eIF4G/4A stabilizes a high-affinity RNA-binding state of eIF4F and enables eIF4A to adopt a more dynamic interaction with eIF4G. This dynamic conformation may contribute to the ability of eIF4F to rapidly bind and release mRNA during scanning.

Stress-induced perturbations in intracellular amino acids reprogram mRNA translation in osmoadaptation independently of the ISR.

The integrated stress response (ISR) plays a pivotal role in adaptation of translation machinery to cellular stress. Here, we demonstrate an ISR-independent osmoadaptation mechanism involving reprogramming of translation via coordinated but independent actions of mTOR and plasma membrane amino acid transporter SNAT2. This biphasic response entails reduced global protein synthesis and mTOR signaling followed by translation of SNAT2. Induction of SNAT2 leads to accumulation of amino acids and reactivation of mTOR and global protein synthesis, paralleled by partial reversal of the early-phase, stress-induced translatome. We propose SNAT2 functions as a molecular switch between inhibition of protein synthesis and establishment of an osmoadaptive translation program involving the formation of cytoplasmic condensates of SNAT2-regulated RNA-binding proteins DDX3X and FUS. In summary, we define key roles of SNAT2 in osmotolerance.

eIF2A-knockout mice reveal decreased life span and metabolic syndrome.

Eukaryotic initiation factor 2A (eIF2A) is a 65 kDa protein that functions in minor initiation pathways, which affect the translation of only a subset of messenger ribonucleic acid (mRNAs), such as internal ribosome entry site (IRES)-containing mRNAs and/or mRNAs harboring upstream near cognate/non-AUG start codons. These non-canonical initiation events are important for regulation of protein synthesis during cellular development and/or the integrated stress response. Selective eIF2A knockdown in cellular systems significantly inhibits translation of such mRNAs, which rely on alternative initiation mechanisms for their translation. However, there exists a gap in our understanding of how eIF2A functions in mammalian systems in vivo (on the organismal level) and ex vivo (in cells). Here, using an eIF2A-knockout (KO) mouse model, we present evidence implicating eIF2A in the biology of aging, metabolic syndrome and central tolerance. We discovered that eIF2A-KO mice have reduced life span and that eIF2A plays an important role in maintenance of lipid homeostasis, the control of glucose tolerance, insulin resistance and also reduces the abundance of B lymphocytes and dendritic cells in the thymic medulla of mice. We also show the eIF2A KO affects male and female mice differently, suggesting that eIF2A may affect sex-specific pathways. Interestingly, our experiments involving pharmacological induction of endoplasmic reticulum (ER) stress with tunicamycin did not reveal any substantial difference between the response to ER stress in eIF2A-KO and wild-type mice. The identification of eIF2A function in the development of metabolic syndrome bears promise for the further identification of specific eIF2A targets responsible for these changes.

Adaptive translational pausing is a hallmark of the cellular response to severe environmental stress.

To survive, mammalian cells must adapt to environmental challenges. While the cellular response to mild stress has been widely studied, how cells respond to severe stress remains unclear. We show here that under severe hyperosmotic stress, cells enter a transient hibernation-like state in anticipation of recovery. We demonstrate this adaptive pausing response (APR) is a coordinated cellular response that limits ATP supply and consumption through mitochondrial fragmentation and widespread pausing of mRNA translation. This pausing is accomplished by ribosome stalling at translation initiation codons, which keeps mRNAs poised to resume translation upon recovery. We further show that recovery from severe stress involves ISR (integrated stress response) signaling that permits cell cycle progression, resumption of growth, and reversal of mitochondria fragmentation. Our findings indicate that cells can respond to severe stress via a hibernation-like mechanism that preserves vital elements of cellular function under harsh environmental conditions.

A Retrospective on eIF2A-and Not the Alpha Subunit of eIF2.

Initiation of protein synthesis in eukaryotes is a complex process requiring more than 12 different initiation factors, comprising over 30 polypeptide chains. The functions of many of these factors have been established in great detail; however, the precise role of some of them and their mechanism of action is still not well understood. Eukaryotic initiation factor 2A (eIF2A) is a single chain 65 kDa protein that was initially believed to serve as the functional homologue of prokaryotic IF2, since eIF2A and IF2 catalyze biochemically similar reactions, i.e., they stimulate initiator Met-tRNAi binding to the small ribosomal subunit. However, subsequent identification of a heterotrimeric 126 kDa factor, eIF2 (α,ß,γ) showed that this factor, and not eIF2A, was primarily responsible for the binding of Met-tRNAi to 40S subunit in eukaryotes. It was found however, that eIF2A can promote recruitment of Met-tRNAi to 40S/mRNA complexes under conditions of inhibition of eIF2 activity (eIF2α-phosphorylation), or its absence. eIF2A does not function in major steps in the initiation process, but is suggested to act at some minor/alternative initiation events such as re-initiation, internal initiation, or non-AUG initiation, important for translational control of specific mRNAs. This review summarizes our current understanding of the eIF2A structure and function.

Rocaglates Induce Gain-of-Function Alterations to eIF4A and eIF4F.

Rocaglates are a diverse family of biologically active molecules that have gained tremendous interest in recent years due to their promising activities in pre-clinical cancer studies. As a result, this family of compounds has been significantly expanded through the development of efficient synthetic schemes. However, it is unknown whether all of the members of the rocaglate family act through similar mechanisms of action. Here, we present a comprehensive study comparing the biological activities of >200 rocaglates to better understand how the presence of different chemical entities influences their biological activities. Through this, we find that most rocaglates preferentially repress the translation of mRNAs containing purine-rich 5' leaders, but certain rocaglates lack this bias in translation repression. We also uncover an aspect of rocaglate mechanism of action in which the pool of translationally active eIF4F is diminished due to the sequestration of the complex onto RNA.

Protein Synthesis Initiation in Eukaryotic Cells.

This review summarizes our current understanding of the major pathway for the initiation phase of protein synthesis in eukaryotic cells, with a focus on recent advances. We describe the major scanning or messenger RNA (mRNA) m7G cap-dependent mechanism, which is a highly coordinated and stepwise regulated process that requires the combined action of at least 12 distinct translation factors with initiator transfer RNA (tRNA), ribosomes, and mRNAs. We limit our review to studies involving either mammalian or budding yeast cells and factors, as these represent the two best-studied experimental systems, and only include a reference to other organisms where particular insight has been gained. We close with a brief description of what we feel are some of the major unknowns in eukaryotic initiation.

A Unique ISR Program Determines Cellular Responses to Chronic Stress.

The integrated stress response (ISR) is a homeostatic mechanism induced by endoplasmic reticulum (ER) stress. In acute/transient ER stress, decreased global protein synthesis and increased uORF mRNA translation are followed by normalization of protein synthesis. Here, we report a dramatically different response during chronic ER stress. This chronic ISR program is characterized by persistently elevated uORF mRNA translation and concurrent gene expression reprogramming, which permits simultaneous stress sensing and proteostasis. The program includes PERK-dependent switching to an eIF3-dependent translation initiation mechanism, resulting in partial, but not complete, translational recovery, which, together with transcriptional reprogramming, selectively bolsters expression of proteins with ER functions. Coordination of transcriptional and translational reprogramming prevents ER dysfunction and inhibits "foamy cell" development, thus establishing a molecular basis for understanding human diseases associated with ER dysfunction.

The Celebration of 40 years of structural biology at Aarhus University as seen through the eyes of a translationalist.

Over the past 50 years, both Brian Clark and I have worked on tRNA, Brian more successfully than I. Our common interests have established collaborations and the training of Aarhus University students in my laboratory. As part of my rejuvenation plan, I have returned to Aarhus and participated in teaching a course in protein synthesis about every five years, beginning in 1990. It has been through this close contact with both Brian and the numerous faculty of the structural biology group that I have been able to witness and appreciate the great strides they have made, both in building a world class department and in the ground breaking discoveries that went with them. While much of the success of the Department of Molecular Biology and Genetics reflects the individual accomplishments of many, the growth of the department is largely a reflection of the dogged determination of Brian to develop one of the most modern and competitive departments in Denmark, if not in Europe (sort of reminds one of Winston Churchill). This retrospective highlights our parallel paths in tRNA and protein synthesis research and Brian's influence in establishing world renowned research in Aarhus.

The eIF2A knockout mouse.

Eukaryotic initiation factor 2A (eIF2A) is a 65-kDa protein that was first identified in the early 1970s as a factor capable of stimulating initiator methionyl-tRNAi (Met-tRNAMeti) binding to 40S ribosomal subunits in vitro. However, in contrast to the eIF2, which stimulates Met-tRNAMeti binding to 40S ribosomal subunits in a GTP-dependent manner, eIF2A didn't reveal any GTP-dependence, but instead was found to direct binding of the Met-tRNAMeti to 40S ribosomal subunits in a codon-dependent manner. eIF2A appears to be highly conserved across eukaryotic species, suggesting conservation of function in evolution. The yeast Saccharomyces cerevisae eIF2A null mutant revealed no apparent phenotype, however, it was found that in yeast eIF2A functions as a suppressor of internal ribosome entry site (IRES)-mediated translation. It was thus suggested that eIF2A my act by impinging on the expression of specific mRNAs. Subsequent studies in mammalian cell systems implicated eIF2A in non-canonical (non-AUG-dependent) translation initiation events involving near cognate UUG and CUG codons. Yet, the role of eIF2A in cellular functions remains largely enigmatic. As a first step toward characterization of the eIF2A function in mammalian systems in vivo, we have obtained homozygous eIF2A-total knockout (KO) mice, in which a gene trap cassette was inserted between eIF2A exons 1 and 2 disrupting expression of all exons downstream of the insertion. The KO mice strain is viable and to date displays no apparent phenotype. We believe that the eIF2A KO mice strain will serve as a valuable tool for researchers studying non-canonical initiation of translation in vivo.

Ternatin and improved synthetic variants kill cancer cells by targeting the elongation factor-1A ternary complex.

Cyclic peptide natural products have evolved to exploit diverse protein targets, many of which control essential cellular processes. Inspired by a series of cyclic peptides with partially elucidated structures, we designed synthetic variants of ternatin, a cytotoxic and anti-adipogenic natural product whose molecular mode of action was unknown. The new ternatin variants are cytotoxic toward cancer cells, with up to 500-fold greater potency than ternatin itself. Using a ternatin photo-affinity probe, we identify the translation elongation factor-1A ternary complex (eEF1A·GTP·aminoacyl-tRNA) as a specific target and demonstrate competitive binding by the unrelated natural products, didemnin and cytotrienin. Mutations in domain III of eEF1A prevent ternatin binding and confer resistance to its cytotoxic effects, implicating the adjacent hydrophobic surface as a functional hot spot for eEF1A modulation. We conclude that the eukaryotic elongation factor-1A and its ternary complex with GTP and aminoacyl-tRNA are common targets for the evolution of cytotoxic natural products.

eIF4F: a retrospective.

The original purification of the heterotrimeric eIF4F was published over 30 years ago (Grifo, J. A., Tahara, S. M., Morgan, M. A., Shatkin, A. J., and Merrick, W. C. (1983) J. Biol. Chem. 258, 5804-5810). Since that time, numerous studies have been performed with the three proteins specifically required for the translation initiation of natural mRNAs, eIF4A, eIF4B, and eIF4F. These have involved enzymatic and structural studies of the proteins and a number of site-directed mutagenesis studies. The regulation of translation exhibited through the mammalian target of rapamycin (mTOR) pathway is predominately seen as the phosphorylation of 4E-BP, an inhibitor of protein synthesis that functions by binding to the cap binding subunit of eIF4F (eIF4E). A hypothesis that requires the disassembly of eIF4F during translation initiation to yield free subunits (eIF4A, eIF4E, and eIF4G) is presented.

OLA1 regulates protein synthesis and integrated stress response by inhibiting eIF2 ternary complex formation.

Translation is a fundamental cellular process, and its dysregulation can contribute to human diseases such as cancer. During translation initiation the eukaryotic initiation factor 2 (eIF2) forms a ternary complex (TC) with GTP and the initiator methionyl-tRNA (tRNAi), mediating ribosomal recruitment of tRNAi. Limiting TC availability is a central mechanism for triggering the integrated stress response (ISR), which suppresses global translation in response to various cellular stresses, but induces specific proteins such as ATF4. This study shows that OLA1, a member of the ancient Obg family of GTPases, is an eIF2-regulatory protein that inhibits protein synthesis and promotes ISR by binding eIF2, hydrolyzing GTP, and interfering with TC formation. OLA1 thus represents a novel mechanism of translational control affecting de novo TC formation, different from the traditional model in which phosphorylation of eIF2α blocks the regeneration of TC. Depletion of OLA1 caused a hypoactive ISR and greater survival in stressed cells. In vivo, OLA1-knockdown rendered cancer cells deficient in ISR and the downstream proapoptotic effector, CHOP, promoting tumor growth and metastasis. Our work suggests that OLA1 is a novel translational GTPase and plays a suppressive role in translation and cell survival, as well as cancer growth and progression.

Rapid kinetics of iron responsive element (IRE) RNA/iron regulatory protein 1 and IRE-RNA/eIF4F complexes respond differently to metal ions.

Metal ion binding was previously shown to destabilize IRE-RNA/IRP1 equilibria and enhanced IRE-RNA/eIF4F equilibria. In order to understand the relative importance of kinetics and stability, we now report rapid rates of protein/RNA complex assembly and dissociation for two IRE-RNAs with IRP1, and quantitatively different metal ion response kinetics that coincide with the different iron responses in vivo. kon, for FRT IRE-RNA binding to IRP1 was eight times faster than ACO2 IRE-RNA. Mn(2+) decreased kon and increased koff for IRP1 binding to both FRT and ACO2 IRE-RNA, with a larger effect for FRT IRE-RNA. In order to further understand IRE-mRNA regulation in terms of kinetics and stability, eIF4F kinetics with FRT IRE-RNA were determined. kon for eIF4F binding to FRT IRE-RNA in the absence of metal ions was 5-times slower than the IRP1 binding to FRT IRE-RNA. Mn(2+) increased the association rate for eIF4F binding to FRT IRE-RNA, so that at 50 µM Mn(2+) eIF4F bound more than 3-times faster than IRP1. IRP1/IRE-RNA complex has a much shorter life-time than the eIF4F/IRE-RNA complex, which suggests that both rate of assembly and stability of the complexes are important, and that allows this regulatory system to respond rapidly to change in cellular iron.

Translational control during endoplasmic reticulum stress beyond phosphorylation of the translation initiation factor eIF2α.

The accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) causes stress to which an unfolded protein response is activated to render cell survival or apoptosis (chronic stress). Transcriptional and translational reprogramming is tightly regulated during the unfolded protein response to ensure specific gene expression. The master regulator of this response is the PERK/eIF2α/ATF4 signaling where eIF2α is phosphorylated (eIF2α-P) by the kinase PERK. This signal leads to global translational shutdown, but it also enables translation of the transcription factor ATF4 mRNA. We showed recently that ATF4 induces an anabolic program through the up-regulation of selected amino acid transporters and aminoacyl-tRNA synthetases. Paradoxically, this anabolic program led cells to apoptosis during chronic ER stress in a manner that involved recovery from stress-induced protein synthesis inhibition. By using eIF2α-P-deficient cells as an experimental system, we identified a communicating network of signaling pathways that contribute to the inhibition of protein synthesis during chronic ER stress. This eIF2α-P-independent network includes (i) inhibition of mammalian target of rapamycin kinase protein complex 1 (mTORC1)-targeted protein phosphorylation, (ii) inhibited translation of a selective group of 5'-terminal oligopyrimidine mRNAs (encoding proteins involved in the translation machinery and translationally controlled by mTORC1 signaling), and (iii) inhibited translation of non-5'-terminal oligopyrimidine ribosomal protein mRNAs and ribosomal RNA biogenesis. We propose that the PERK/eIF2α-P/ATF4 signaling acts as a brake in the decline of protein synthesis during chronic ER stress by positively regulating signaling downstream of the mTORC1 activity. These studies advance our knowledge on the complexity of the communicating signaling pathways in controlling protein synthesis rates during chronic stress.

Control not at initiation? Bah, humbug!

Initiation is often assumed to be rate-limiting for protein synthesis, but the presence of rare codons nonetheless can influence protein levels. In this issue of The EMBO Journal, Chu et al report that when rare codons are positioned near the start of the coding region, 'liberation' of the initiation codon for loading of the next 40S subunit may be rate­limiting for initiation and therefore overall protein synthesis. The sequential nature of translation results in an interdependence in ribosome association either by de novo initiation or recycling. Thus, a more general view emerges where both elongation and initiation can contribute to protein expression.

Influence of translation factor activities on start site selection in six different mRNAs.

Current literature using biochemical assays, structural analyses and genetic manipulations has reported that the key factors associated with the faithful matching of the initiator met-tRNA to the start codon AUG are eIF1, eIF1A and eIF5. However, these findings were in each case based upon the utilization of a single mRNA, perhaps with variations. In an effort to evaluate this general finding, we tested six different mRNAs. Our results confirm that these three proteins are important for start site selection. However, two additional findings would not have been predicted. The first is that eIF1 plays a major role in selecting against start codons that are in close proximity to the 5' end of the mRNA (i.e., less than 21 nucleotides). Second, the addition of eIF5B had nearly the same affect as the addition of eIF5. This is unexpected given the different roles that eIF5 and eIF5B have been proposed to play in the 80S initiation pathway. Finally, although many of the mRNAs appear to respond qualitatively in a similar manner, the quantitative differences noted suggest that there is still some mRNA specific character to our findings. This character may be the length of the 5' UTR, involvement of an IRES element, secondary structure either 5' or 3' of the start codon or specific sequence/structure elements that interact with RNA binding proteins or the ribosome.

Fe2+ binds iron responsive element-RNA, selectively changing protein-binding affinities and regulating mRNA repression and activation.

Iron increases synthesis rates of proteins encoded in iron-responsive element (IRE)-mRNAs; metabolic iron ("free," "labile") is Fe(2+). The noncoding IRE-RNA structure, approximately 30 nt, folds into a stem loop to control synthesis of proteins in iron trafficking, cell cycling, and nervous system function. IRE-RNA riboregulators bind specifically to iron-regulatory proteins (IRP) proteins, inhibiting ribosome binding. Deletion of the IRE-RNA from an mRNA decreases both IRP binding and IRP-independent protein synthesis, indicating effects of other "factors." Current models of IRE-mRNA regulation, emphasizing iron-dependent degradation/modification of IRP, lack answers about how iron increases IRE-RNA/IRP protein dissociation or how IRE-RNA, after IRP dissociation, influences protein synthesis rates. However, we observed Fe(2+) (anaerobic) or Mn(2+) selectively increase the IRE-RNA/IRP K(D). Here we show: (i) Fe(2+) binds to the IRE-RNA, altering its conformation (by 2-aminopurine fluorescence and ethidium bromide displacement); (ii) metal ions increase translation of IRE-mRNA in vitro; (iii) eukaryotic initiation factor (eIF)4F binds specifically with high affinity to IRE-RNA; (iv) Fe(2+) increased eIF4F/IRE-RNA binding, which outcompetes IRP binding; (v) exogenous eIF4F rescued metal-dependent IRE-RNA translation in eIF4F-depeleted extracts. The regulation by metabolic iron binding to IRE-RNA to decrease inhibitor protein (IRP) binding and increase activator protein (eIF4F) binding identifies IRE-RNA as a riboregulator.

Chimeric glutathione S-transferases containing inserts of kininogen peptides: potential novel protein therapeutics.

The study of synthetic peptides corresponding to discrete regions of proteins has facilitated the understanding of protein structure-activity relationships. Short peptides can also be used as powerful therapeutic agents. However, in many instances, small peptides are prone to rapid degradation or aggregation and may lack the conformation required to mimic the functional motifs of the protein. For peptides to function as pharmacologically active agents, efficient production or expression, high solubility, and retention of biological activity through purification and storage steps are required. We report here the design, expression, and functional analysis of eight engineered GST proteins (denoted GSHKTs) in which peptides ranging in size from 8 to 16 amino acids and derived from human high molecular weight kininogen (HK) domain 5 were inserted into GST (between Gly-49 and Leu-50). Peptides derived from HK are known to inhibit cell proliferation, angiogenesis, and tumor metastasis, and the biological activity of the HK peptides was dramatically (>50-fold) enhanced following insertion into GST. GSHKTs are soluble and easily purified from Escherichia coli by affinity chromatography. Functionally, these hybrid proteins cause inhibition of endothelial cell proliferation. Crystallographic analysis of GSHKT10 and GSHKT13 (harboring 10- and 13-residue HK peptides, respectively) showed that the overall GST structure was not perturbed. These results suggest that the therapeutic efficacy of short peptides can be enhanced by insertion into larger proteins that are easily expressed and purified and that GST may potentially be used as such a carrier.

A new framework for understanding IRES-mediated translation.

Studies over the past 5 or so years have indicated that the traditional clustering of mechanisms for translation initiation in eukaryotes into cap-dependent and cap-independent (or IRES-mediated) is far too narrow. From individual studies of a number of mRNAs encoding proteins that are regulatory in nature (i.e. likely to be needed in small amounts such as transcription factors, protein kinases, etc.), it is now evident that mRNAs exist that blur these boundaries. This review seeks to set the basic ground rules for the analysis of different initiation pathways that are associated with these new mRNAs as well as related to the more traditional mechanisms, especially the cap-dependent translational process that is the major route of initiation of mRNAs for housekeeping proteins and thus, the bulk of protein synthesis in most cells. It will become apparent that a mixture of descriptions is likely to become the norm in the near future (i.e. m(7)G-assisted internal initiation).