Cytosolic and mitochondrial translation elongation are coordinated through the molecular chaperone TRAP1 for the synthesis and import of mitochondrial proteins.

A complex interplay between mRNA translation and cellular respiration has been recently unveiled, but its regulation in humans is poorly characterized in either health or disease. Cancer cells radically reshape both biosynthetic and bioenergetic pathways to sustain their aberrant growth rates. In this regard, we have shown that the molecular chaperone TRAP1 not only regulates the activity of respiratory complexes, behaving alternatively as an oncogene or a tumor suppressor, but also plays a concomitant moonlighting function in mRNA translation regulation. Herein, we identify the molecular mechanisms involved, showing that TRAP1 (1) binds both mitochondrial and cytosolic ribosomes, as well as translation elongation factors; (2) slows down translation elongation rate; and (3) favors localized translation in the proximity of mitochondria. We also provide evidence that TRAP1 is coexpressed in human tissues with the mitochondrial translational machinery, which is responsible for the synthesis of respiratory complex proteins. Altogether, our results show an unprecedented level of complexity in the regulation of cancer cell metabolism, strongly suggesting the existence of a tight feedback loop between protein synthesis and energy metabolism, based on the demonstration that a single molecular chaperone plays a role in both mitochondrial and cytosolic translation, as well as in mitochondrial respiration.

Proteomic analysis of nascent polypeptide chains that potentially induce translational pausing during elongation.

Currently, proteins equipped with "ribosomal arrest peptides" (RAPs) that regulate the expression of downstream genes and their own activity by pausing their own translation during elongation are extensively studied. However, studies focusing on RAP have been conducted primarily in prokaryotic cells; studies on eukaryotic cells, especially mammalian cells, are limited. In the present study, we comprehensively examined translationally arrested nascent polypeptides to gain novel insights into RAPs in mammalian cells. Cetyltrimethylammonium bromide was used to obtain nascent polypeptide chains that were translationally arrested during translation elongation. After proteomic analysis, additional screening by discriminating according to amino acid residues at the C-terminal end revealed several novel RAP candidates. Our method can be applied for comprehensive RAP studies in mammalian cells.

A functional connection between translation elongation and protein folding at the ribosome exit tunnel in Saccharomyces cerevisiae.

Proteostasis needs to be tightly controlled to meet the cellular demand for correctly de novo folded proteins and to avoid protein aggregation. While a coupling between translation rate and co-translational folding, likely involving an interplay between the ribosome and its associated chaperones, clearly appears to exist, the underlying mechanisms and the contribution of ribosomal proteins remain to be explored. The ribosomal protein uL3 contains a long internal loop whose tip region is in close proximity to the ribosomal peptidyl transferase center. Intriguingly, the rpl3[W255C] allele, in which the residue making the closest contact to this catalytic site is mutated, affects diverse aspects of ribosome biogenesis and function. Here, we have uncovered, by performing a synthetic lethal screen with this allele, an unexpected link between translation and the folding of nascent proteins by the ribosome-associated Ssb-RAC chaperone system. Our results reveal that uL3 and Ssb-RAC cooperate to prevent 80S ribosomes from piling up within the 5' region of mRNAs early on during translation elongation. Together, our study provides compelling in vivo evidence for a functional connection between peptide bond formation at the peptidyl transferase center and chaperone-assisted de novo folding of nascent polypeptides at the solvent-side of the peptide exit tunnel.

Natural products targeting the elongation phase of eukaryotic protein biosynthesis.

Covering: 2000 to 2020 The translation of mRNA into proteins is a precisely regulated, complex process that can be divided into three main stages, i.e. initiation, elongation, termination, and recycling. This contribution is intended to highlight how natural products interfere with the elongation phase of eukaryotic protein biosynthesis. Cycloheximide, isolated from Streptomyces griseus, has long been the prototype inhibitor of eukaryotic translation elongation. In the last three decades, a variety of natural products from different origins were discovered to also address the elongation step in different manners, including interference with the elongation factors eEF1 and eEF2 as well as binding to A-, P- or E-sites of the ribosome itself. Recent advances in the crystallization of the ribosomal machinery together with natural product inhibitors allowed characterizing similarities as well as differences in their mode of action. Since aberrations in protein synthesis are commonly observed in tumors, and malfunction or overexpression of translation factors can cause cellular transformation, the protein synthesis machinery has been realized as an attractive target for anticancer drugs. The therapeutic use of the first natural products that reached market approval, plitidepsin (Aplidin®) and homoharringtonine (Synribo®), will be introduced. In addition, we will highlight two other potential indications for translation elongation inhibitors, i.e. viral infections and genetic disorders caused by premature termination of translation.

Structural and functional studies revealed key mechanisms underlying elongation step of protein translation.

The ribosome is an ancient and universally conserved macromolecular machine that synthesizes proteins in all organisms. Since the discovery of the ribosome by electron microscopy in the mid-1950s, rapid progress has been made in research on it, regarding its architecture and functions. As a machine that synthesizes polypeptides, the sequential addition of amino acids to a growing polypeptide chain occurs during a phase called the elongation cycle. This is the core step of protein translation and is highly conserved between bacteria and eukarya. The elongation cycle involves codon recognition by aminoacyl tRNAs, catalysis of peptide bond formation, and the most complex operation of translation-translocation. In this review, we discuss the fundamental results from structural and functional studies over the past decades that have led to understanding of the three key questions underlying translation.

Nascent Polypeptide Domain Topology and Elongation Rate Direct the Cotranslational Hierarchy of Hsp70 and TRiC/CCT.

Cotranslational protein folding requires assistance from elaborate ribosome-associated chaperone networks. It remains unclear how the changing information in a growing nascent polypeptide dictates the recruitment of functionally distinct chaperones. Here, we used ribosome profiling to define the principles governing the cotranslational action of the chaperones TRiC/CCT and Hsp70/Ssb. We show that these chaperones are sequentially recruited to specific sites within domain-encoding regions of select nascent polypeptides. Hsp70 associates first, binding select sites throughout domains, whereas TRiC associates later, upon the emergence of nearly complete domains that expose an unprotected hydrophobic surface. This suggests that transient topological properties of nascent folding intermediates drive sequential chaperone association. Moreover, cotranslational recruitment of both TRiC and Hsp70 correlated with translation elongation slowdowns. We propose that the temporal modulation of the nascent chain structural landscape is coordinated with local elongation rates to regulate the hierarchical action of Hsp70 and TRiC for cotranslational folding.

Elongation factor P is required to maintain proteome homeostasis at high growth rate.

Elongation factor P (EF-P) is a universally conserved translation factor that alleviates ribosome pausing at polyproline (PPX) motifs by facilitating peptide bond formation. In the absence of EF-P, PPX peptide bond formation can limit translation rate, leading to pleotropic phenotypes including slowed growth, increased antibiotic sensitivity, and loss of virulence. In this study, we observe that many of these phenotypes are dependent on growth rate. Limiting growth rate suppresses a variety of detrimental phenotypes associated with ribosome pausing at PPX motifs in the absence of EF-P. Polysome levels are also similar to wild-type under slow growth conditions, consistent with global changes in ribosome queuing in cells without EF-P when growth rate is decreased. Inversely, under high protein synthesis demands, we observe that Escherichia coli lacking EF-P have reduced fitness. Our data demonstrate that EF-P-mediated relief of ribosome queuing is required to maintain proteome homeostasis under conditions of high translational demands.

The expression profile and prognostic significance of eukaryotic translation elongation factors in different cancers.

Eukaryotic translation factors, especially initiation factors have garnered much attention with regards to their role in the onset and progression of different cancers. However, the expression levels and prognostic significance of translation elongation factors remain poorly explored in different cancers. In this study, we have investigated the mRNA transcript levels of seven translation elongation factors in different cancer types using Oncomine and TCGA databases. Furthermore, we have identified the prognostic significance of these factors using Kaplan-Meier Plotter and SurvExpress databases. We observed altered expression levels of all the elongation factors in different cancers. Higher expression of EEF1A2, EEF1B2, EEF1G, EEF1D, EEF1E1 and EEF2 was observed in most of the cancer types, whereas reverse trend was observed for EEF1A1. Overexpression of many factors predicted poor prognosis in breast (EEF1D, EEF1E1, EEF2) and lung cancer (EEF1A2, EEF1B2, EEF1G, EEF1E1). However, we didn't see any common correlation of expression levels of elongation factors with survival outcomes across cancer types. Cancer subtype stratification showed association of survival outcomes and expression levels of elongation factors in specific sub-types of breast, lung and gastric cancer. Most interestingly, we observed a reciprocal relationship between the expression levels of the two EEF1A isoforms viz. EEF1A1 and EEF1A2, in most of the cancer types. Our results suggest that translation elongation factors can have a role in tumorigenesis and affect survival in cancer specific manner. Elongation factors have potential to serve as biomarkers and therapeutic drug targets, yet further study is required. Reciprocal relationship of differential expression between EEF1A isoforms observed in multiple cancer types indicates opposing roles in cancer and needs further investigation.

Heat shock protein 70 promotes coxsackievirus B3 translation initiation and elongation via Akt-mTORC1 pathway depending on activation of p70S6K and Cdc2.

We previously demonstrated that coxsackievirus B3 (CVB3) infection upregulated heat shock protein 70 (Hsp70) and promoted CVB3 multiplication. Here, we report the underlying mechanism by which Hsp70 enhances viral RNA translation. By using an Hsp70-overexpressing cell line infected with CVB3, we found that Hsp70 enhanced CVB3 VP1 translation at two stages. First, Hsp70 induced upregulation of VP1 translation at the initiation stage via upregulation of internal ribosome entry site trans-acting factor lupus autoantigen protein and activation of eIF4E binding protein 1, a cap-dependent translation suppressor. Second, we found that Hsp70 increased CVB3 VP1 translation by enhancing translation elongation. This was mediated by the Akt-mammalian target of rapamycin complex 1 signal cascade, which led to the activation of eukaryotic elongation factor 2 via p70S6K- and cell division cycle protein 2 homolog (Cdc2)-mediated phosphorylation and inactivation of eukaryotic elongation factor 2 kinase. We also determined the position of Cdc2 in this signal pathway, indicating that Cdc2 is regulated by mammalian target of rapamycin complex 1. This signal transduction pathway was validated using a number of specific pharmacological inhibitors, short interfering RNAs (siRNAs) and a dominant negative Akt plasmid. Because Hsp70 is a central component of the cellular network of molecular chaperones enhancing viral replication, these data may provide new strategies to limit this viral infection.

Protein Elongation, Co-translational Folding and Targeting.

The elongation phase of protein synthesis defines the overall speed and fidelity of protein synthesis and affects protein folding and targeting. The mechanisms of reactions taking place during translation elongation remain important questions in understanding ribosome function. The ribosome-guided by signals in the mRNA-can recode the genetic information, resulting in alternative protein products. Co-translational protein folding and interaction of ribosomes and emerging polypeptides with associated protein biogenesis factors determine the quality and localization of proteins. In this review, we summarize recent findings on mechanisms of translation elongation in bacteria, including decoding and recoding, peptide bond formation, tRNA-mRNA translocation, co-translational protein folding, interaction with protein biogenesis factors and targeting of ribosomes synthesizing membrane proteins to the plasma membrane. The data provide insights into how the ribosome shapes composition and quality of the cellular proteome.

Differential requirement of F-actin and microtubule cytoskeleton in cue-induced local protein synthesis in axonal growth cones.

BACKGROUND: Local protein synthesis (LPS) via receptor-mediated signaling plays a role in the directional responses of axons to extrinsic cues. An intact cytoskeleton is critical to enact these responses, but it is not known whether the two major cytoskeletal elements, F-actin and microtubules, have any roles in regulating axonal protein synthesis. RESULTS: Here, we show that pharmacological disruption of either microtubules or actin filaments in growth cones blocks netrin-1-induced de novo synthesis of proteins, as measured by metabolic incorporation of labeled amino acids, implicating both elements in axonal synthesis. However, comparative analysis of the activated translation initiation regulator, eIF4E-BP1, revealed a striking difference in the point of action of the two elements: actin disruption completely inhibited netrin-1-induced eIF4E-BP1 phosphorylation while microtubule disruption had no effect. An intact F-actin, but not microtubule, cytoskeleton was also required for netrin-1-induced activation of the PI3K/Akt/mTOR pathway, upstream of translation initiation. Downstream of translation initiation, microtubules were required for netrin-1-induced activation of eukaryotic elongation factor 2 kinase (eEF2K) and eEF2. CONCLUSIONS: Taken together, our results show that while actin and microtubules are both crucial for cue-induced axonal protein synthesis, they serve distinct roles with F-actin being required for the initiation of translation and microtubules acting later at the elongation step.

Eukaryotic elongation factor 2 kinase regulates the cold stress response by slowing translation elongation.

Cells respond to external stress conditions by controlling gene expression, a process which occurs rapidly via post-transcriptional regulation at the level of protein synthesis. Global control of translation is mediated by modification of translation factors to allow reprogramming of the translatome and synthesis of specific proteins that are required for stress protection or initiation of apoptosis. In the present study, we have investigated how global protein synthesis rates are regulated upon mild cooling. We demonstrate that although there are changes to the factors that control initiation, including phosphorylation of eukaryotic translation initiation factor 2 (eIF2) on the α-subunit, the reduction in the global translation rate is mediated by regulation of elongation via phosphorylation of eukaryotic elongation factor 2 (eEF2) by its specific kinase, eEF2K (eukaryotic elongation factor 2 kinase). The AMP/ATP ratio increases following cooling, consistent with a reduction in metabolic rates, giving rise to activation of AMPK (5'-AMP-activated protein kinase), which is upstream of eEF2K. However, our data show that the major trigger for activation of eEF2K upon mild cooling is the release of Ca2+ ions from the endoplasmic reticulum (ER) and, importantly, that it is possible to restore protein synthesis rates in cooled cells by inhibition of this pathway at multiple points. As cooling has both therapeutic and industrial applications, our data provide important new insights into how the cellular responses to this stress are regulated, opening up new possibilities to modulate these responses for medical or industrial use at physiological or cooler temperatures.

p53-directed translational control can shape and expand the universe of p53 target genes.

The increasing number of genome-wide transcriptome analyses focusing on p53-induced cellular responses in many cellular contexts keeps adding to the already numerous p53-regulated transcriptional networks. To investigate post-transcriptional controls as an additional dimension of p53-directed gene expression responses, we performed a translatome analysis through polysomal profiling on MCF7 cells upon 16 hours of doxorubicin or nutlin-3a treatment. The comparison between the transcriptome and the translatome revealed a considerable level of uncoupling, characterized by genes whose transcription variations did not correlate with translation variations. Interestingly, uncoupled genes were associated with apoptosis, DNA and RNA metabolism and cell cycle functions, suggesting that post-transcriptional control can modulate classical p53-regulated responses. Furthermore, even for well-established p53 targets that were differentially expressed both at the transcriptional and translational levels, quantitative differences between the transcriptome, subpolysomal and polysomal RNAs were evident. As we searched mechanisms underlying gene expression uncoupling, we identified the p53-dependent modulation of six RNA-binding proteins, where hnRNPD (AUF1) and CPEB4 are direct p53 transcriptional targets, whereas SRSF1, DDX17, YBX1 and TARDBP are indirect targets (genes modulated preferentially in the subpolysomal or polysomal mRNA level) modulated at the translational level in a p53-dependent manner. In particular, YBX1 translation appeared to be reduced by p53 via two different mechanisms, one related to mTOR inhibition and the other to miR-34a expression. Overall, we established p53 as a master regulator of translational control and identified new p53-regulated genes affecting translation that can contribute to p53-dependent cellular responses.

Ibuprofen treatment blunts early translational signaling responses in human skeletal muscle following resistance exercise.

Cyclooxygenase-1 and -2 pathway-derived prostaglandins (PGs) have been implicated in adaptive muscle responses to exercise, but the role of PGs in contraction-induced muscle signaling has not been determined. We investigated the effect of inhibition of cyclooxygenase-1 and -2 activities with the nonsteroidal anti-inflammatory drug ibuprofen on human muscle signaling responses to resistance exercise. Subjects orally ingested 1,200 mg ibuprofen (or placebo control) in three 400-mg doses administered ∼30 min before and ∼6 h and ∼12 h following a bout of unaccustomed resistance exercise (80% one repetition maximum). Muscle biopsies were obtained at rest (preexercise), immediately postexercise (0 h), 3 h postexercise, and at 24 h of recovery. In the placebo (PLA) group, phosphorylation of ERK1/2 (Thr202/Tyr204), ribosomal protein S6 kinase (RSK, Ser380), mitogen-activated kinase 1 (Mnk1, Thr197/202), and p70S6 kinase (p70S6K, Thr421/Ser424) increased at both 0 and 3 h postexercise, with delayed elevation of phospho (p)-p70S6K (Thr389) and p-rpS6 (Ser235/S36 and Ser240/244) at 3 h postexercise. Only p-ERK1/2 (Thr202/Tyr204) remained significantly elevated in the 24-h postexercise biopsy. Ibuprofen treatment prevented sustained elevation of MEK-ERK signaling at 3 h (p-ERK1/2, p-RSK, p-Mnk1, p-p70S6K Thr421/Ser424) and 24 h (p-ERK1/2) postexercise, and this was associated with suppressed phosphorylation of ribosomal protein S6 (Ser235/236 and Ser240/244). Early contraction-induced p-Akt (Ser473) and p-p70S6K (Thr389) were not influenced by ibuprofen, but p-p70S6K (Thr389) remained elevated 24 h postexercise only in those receiving ibuprofen treatment. Early muscle signaling responses to resistance exercise are, in part, ibuprofen sensitive, suggesting that PGs are important signaling molecules during early postexercise recovery.

Initiation with elongator tRNAs.

In all domains of life, initiator tRNA functions exclusively at the first step of protein synthesis while elongator tRNAs extend the polypeptide chain. Unique features of initiator tRNA enable it to preferentially bind the ribosomal P site and initiate translation. Recently, we showed that the abundance of initiator tRNA also contributes to its specialized role. This motivates the question, can a cell also use elongator tRNA to initiate translation under certain conditions? To address this, we introduced non-AUG initiation codons CCC (Pro), GAG (Glu), GGU (Gly), UCU (Ser), UGU (Cys), ACG (Thr), AAU (Asn), and AGA (Arg) into the uracil DNA glycosylase gene (ung) used as a reporter gene. Enzyme assays from log-phase cells revealed initiation from non-AUG codons when intracellular initiator tRNA levels were reduced. The activity increased significantly in stationary phase. Further increases in initiation from non-AUG codons occurred in both growth phases upon introduction of plasmid-borne genes of cognate elongator tRNAs. Since purine-rich Shine-Dalgarno sequences occur frequently on mRNAs (in places other than the canonical AUG codon initiation contexts), initiation with elongator tRNAs from the alternate contexts may generate proteome diversity under stress without compromising genomic integrity. Thus, by changing the relative amounts of initiator and elongator tRNAs within the cell, we have blurred the distinction between the two classes of tRNAs thought to be frozen through years of evolution.

Emi1 preferentially inhibits ubiquitin chain elongation by the anaphase-promoting complex.

The anaphase-promoting complex (APC) is the crucial ubiquitin ligase targeting the regulatory machinery of the cell cycle. Emi1, a major modulator of APC activity, is thought to act competitively as a pseudosubstrate. We show that the modulation of APC activity is more subtle: Emi1 inhibits ubiquitylation at both substrate binding and separately at the step of ubiquitin transfer to APC-bound substrates. The zinc-binding region of Emi1 allows multiple monoubiquitylation of substrates, but preferentially suppresses the ubiquitin chain elongation by UBCH10. Furthermore, the carboxy-terminal tail of Emi1 antagonizes chain elongation by Ube2S, by competitively preventing its binding to the APC cullin subunit through electrostatic interaction. Combinatorially, Emi1 effectively stabilizes APC substrates by suppressing ubiquitin chain extension. Deubiquitylating enzymes can then convert inhibited substrates to their basal state. Chain elongation may be a particularly sensitive step for controlling degradation, and this study provides the first kinetic evidence for how it is inhibited.

(R)-ß-lysine-modified elongation factor P functions in translation elongation.

Post-translational modification of bacterial elongation factor P (EF-P) with (R)-ß-lysine at a conserved lysine residue activates the protein in vivo and increases puromycin reactivity of the ribosome in vitro. The additional hydroxylation of EF-P at the same lysine residue by the YfcM protein has also recently been described. The roles of modified and unmodified EF-P during different steps in translation, and how this correlates to its physiological role in the cell, have recently been linked to the synthesis of polyproline stretches in proteins. Polysome analysis indicated that EF-P functions in translation elongation, rather than initiation as proposed previously. This was further supported by the inability of EF-P to enhance the rate of formation of fMet-Lys or fMet-Phe, indicating that the role of EF-P is not to specifically stimulate formation of the first peptide bond. Investigation of hydroxyl-(ß)-lysyl-EF-P showed 30% increased puromycin reactivity but no differences in dipeptide synthesis rates when compared with the ß-lysylated form. Unlike disruption of the other genes required for EF-P modification, deletion of yfcM had no phenotypic consequences in Salmonella. Taken together, our findings indicate that EF-P functions in translation elongation, a role critically dependent on post-translational ß-lysylation but not hydroxylation.

Translation elongation regulates substrate selection by the signal recognition particle.

The signal recognition particle (SRP) is a universally conserved cellular machinery responsible for delivering membrane and secretory proteins to the proper cellular destination. The precise mechanism by which fidelity is achieved by the SRP pathway within the in vivo environment is yet to be understood. Previous studies have focused on the SRP pathway in isolation. Here we describe another important factor that modulates substrate selection by the SRP pathway: the ongoing synthesis of the nascent polypeptide chain by the ribosome. A slower translation elongation rate rescues the targeting defect of substrate proteins bearing mutant, suboptimal signal sequences both in vitro and in vivo. Consistent with a kinetic origin of this effect, similar rescue of protein targeting was also observed with mutant SRP receptors or SRP RNAs that specifically compromise the kinetics of SRP-receptor interaction during protein targeting. These data are consistent with a model in which ongoing protein translation is in constant kinetic competition with the targeting of the nascent proteins by the SRP and provides an important factor to regulate the fidelity of substrate selection by the SRP.

Identification of an mRNP complex regulating tumorigenesis at the translational elongation step.

Transcript-selective translational regulation of epithelial-mesenchymal transition (EMT) by transforming growth factor-ß (TGF-ß) is directed by the hnRNP E1-containing TGF-ß-activated-translational (BAT) mRNP complex. Herein, eukaryotic elongation factor-1 A1 (eEF1A1) is identified as an integral component of the BAT complex. Translational silencing of Dab2 and ILEI, two EMT transcripts, is mediated by the binding of hnRNP E1 and eEF1A1 to their 3'UTR BAT element, whereby hnRNP E1 stalls translational elongation by inhibiting the release of eEF1A1 from the ribosomal A site. TGF-ß-mediated hnRNP E1 phosphorylation, through Akt2, disrupts the BAT complex, thereby restoring translation of target EMT transcripts. Attenuation of hnRNP E1 expression in two noninvasive breast epithelial cells (NMuMG and MCF-7) not only induced EMT but also enabled cells to form metastatic lesions in vivo. Thus, translational regulation by TGF-ß at the elongation stage represents a critical checkpoint coordinating the expression of EMT transcripts required during development and in tumorigenesis and metastatic progression.