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.

IRC3 Regulates Mitochondrial Translation in Response to Metabolic Cues in Saccharomyces cerevisiae.

Mitochondrial oxidative phosphorylation (OXPHOS) enzymes have a dual genetic origin. Mechanisms regulating the expression of nucleus-encoded OXPHOS subunits in response to metabolic cues (glucose versus glycerol) are well understood, while the regulation of mitochondrially encoded OXPHOS subunits is poorly defined. Here, we show that IRC3, a DEAD/H box helicase gene, previously implicated in mitochondrial DNA maintenance, is central to integrating metabolic cues with mitochondrial translation. Irc3 associates with mitochondrial small ribosomal subunits in cells consistent with its role in regulating translation elongation based on the Arg8m reporter system. IRC3-deleted cells retained mitochondrial DNA despite a growth defect on glycerol plates. Glucose-grown Δirc3ρ+ and irc3 temperature-sensitive cells at 37°C have reduced translation rates from the majority of mRNAs. In contrast, when galactose was the carbon source, a reduction in mitochondrial translation was observed predominantly from Cox1 mRNA in Δirc3ρ+ cells but no defect was observed in irc3 temperature-sensitive cells, at 37°C. In support of a model whereby IRC3 responds to metabolic cues to regulate mitochondrial translation, Δirc3 suppressor strains isolated for restoration of growth on glycerol medium restore mitochondrial protein synthesis differentially in the presence of glucose versus glycerol.

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.

Dynamics of uS19 C-Terminal Tail during the Translation Elongation Cycle in Human Ribosomes.

Ribosomes undergo multiple conformational transitions during translation elongation. Here, we report the high-resolution cryoelectron microscopy (cryo-EM) structure of the human 80S ribosome in the post-decoding pre-translocation state (classical-PRE) at 3.3-Å resolution along with the rotated (hybrid-PRE) and the post-translocation states (POST). The classical-PRE state ribosome structure reveals a previously unobserved interaction between the C-terminal region of the conserved ribosomal protein uS19 and the A- and P-site tRNAs and the mRNA in the decoding site. In addition to changes in the inter-subunit bridges, analysis of different ribosomal conformations reveals the dynamic nature of this domain and suggests a role in tRNA accommodation and translocation during elongation. Furthermore, we show that disease-associated mutations in uS19 result in increased frameshifting. Together, this structure-function analysis provides mechanistic insights into the role of the uS19 C-terminal tail in the context of mammalian ribosomes.

Translation elongation factor P (EF-P).

Translation elongation factor P (EF-P) is conserved in all three domains of life (called eIF5A and aIF5A in eukaryotes and archaea, respectively) and functions to alleviate ribosome pausing during the translation of specific sequences, including consecutive proline residues. EF-P was identified in 1975 as a factor that stimulated the peptidyltransferase reaction in vitro but its involvement in the translation of tandem proline residues was not uncovered until 2013. Throughout the four decades of EF-P research, perceptions of EF-P function have changed dramatically. In particular, while EF-P was thought to potentiate the formation of the first peptide bond in a protein, it is now broadly accepted to act throughout translation elongation. Further, EF-P was initially reported to be essential, but recent work has shown that the requirement of EF-P for growth is conditional. Finally, it is thought that post-translational modification of EF-P is strictly required for its function but recent studies suggest that EF-P modification may play a more nuanced role in EF-P activity. Here, we review the history of EF-P research, with an emphasis on its initial isolation and characterization as well as the discoveries that altered our perceptions of its function.

Mitochondrial mRNA fragments are circularized in a human HEK cell line.

The relatively recent focus on the widespread occurrence and abundance of circular RNAs (circRNA) in the human cell nucleus has sparked an intensive interest in their existence and possible roles in cell gene expression and physiology. The presence of circRNAs in mammalian mitochondria, however, has been under-explored. Mitochondrial mRNAs differ from those produced from nuclear genes because they lack introns and are transcribed as poly-cistronic transcripts that are endonucleolytically cleaved, leaving transcripts with very small 5' and 3' UTRs. Circular RNAs have been identified in the semi-autonomous organelles of single-celled organisms and plants but their purpose has not been clearly demonstrated. The goal of our project was to test the hypothesis, processed mRNAs are circularized in vertebrate mitochondria as a necessary RNA processing step prior to translation. Mitochondrial mRNAs were isolated from the human cell line HEK293 and evidence of circularization sought by treating RNA with RNAse-R and then amplifying putative 3'-5' junction sites. Sequence results demonstrated the occurrence of mRNA circularization within each coding region of the mitochondrial genome. However, in most cases the circRNAs carried coding regions that had been truncated, suggesting they were not translatable. Quantification of the circularized versions of the mRNAs revealed they comprise a small portion (~10%) of the total mRNA. These findings demonstrate that mRNA circularization occurs in mammalian mitochondria but it does not appear to play a role in making translatable mRNAs.

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.

Molecular mechanisms relating to amino acid regulation of protein synthesis.

Some amino acids (AA) act through several signalling pathways and mechanisms to mediate the control of gene expression at the translation level, and the regulation occurs, specifically, on the initiation and the signalling pathways for translation. The translation of mRNA to protein synthesis proceeds through the steps of initiation and elongation, and AA act as important feed-forward activators that are involved in many pathways, such as the sensing and the transportation of AA by cells, in these steps in many tissues of mammals. For the translation, phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) is a critical molecule that controls the translation initiation and its functions can be regulated by some AA. Another control point in the mRNA binding step in the translation initiation is at the regulation by mammalian target of rapamycin, which requires a change of phosphorylation status of ribosomal protein S6. In fact, the change of phosphorylation status of ribosomal protein S6 might be involved in global protein synthesis. The present review summarises recent work on the molecular mechanisms of the regulation of protein synthesis by AA and highlights new findings.

Novel insights into gene expression regulation during meiosis revealed by translation elongation dynamics.

The ability to dynamically control mRNA translation has a great impact on many intracellular processes. Whereas it is believed that translational control in eukaryotes occurs mainly at initiation, the condition-specific changes at the elongation level and their potential regulatory role remain unclear. Using computational approaches applied to ribosome profiling data, we show that elongation rate is dynamic and can change considerably during the yeast meiosis to facilitate the selective translation of stage-specific transcripts. We observed unique elongation changes during meiosis II, including a global inhibition of translation elongation at the onset of anaphase II accompanied by a sharp shift toward increased elongation for genes required at this meiotic stage. We also show that ribosomal proteins counteract the global decreased elongation by maintaining high initiation rates. Our findings provide new insights into gene expression regulation during meiosis and demonstrate that codon usage evolved, among others, to optimize timely translation.

Ena/VASP processive elongation is modulated by avidity on actin filaments bundled by the filopodia cross-linker fascin.

Ena/VASP tetramers are processive actin elongation factors that localize to diverse F-actin networks composed of filaments bundled by different cross-linking proteins, such as filopodia (fascin), lamellipodia (fimbrin), and stress fibers (α-actinin). Previously, we found that Ena takes approximately threefold longer processive runs on trailing barbed ends of fascin-bundled F-actin. Here, we used single-molecule TIRFM (total internal reflection fluorescence microscopy) and developed a kinetic model to further dissect Ena/VASP's processive mechanism on bundled filaments. We discovered that Ena's enhanced processivity on trailing barbed ends is specific to fascin bundles, with no enhancement on fimbrin or α-actinin bundles. Notably, Ena/VASP's processive run length increases with the number of both fascin-bundled filaments and Ena "arms," revealing avidity facilitates enhanced processivity. Consistently, Ena tetramers form more filopodia than mutant dimer and trimers in Drosophila culture cells. Moreover, enhanced processivity on trailing barbed ends of fascin-bundled filaments is an evolutionarily conserved property of Ena/VASP homologues, including human VASP and Caenorhabditis elegans UNC-34. These results demonstrate that Ena tetramers are tailored for enhanced processivity on fascin bundles and that avidity of multiple arms associating with multiple filaments is critical for this process. Furthermore, we discovered a novel regulatory process whereby bundle size and bundling protein specificity control activities of a processive assembly factor.

Ribosomal protein eL42 contributes to the catalytic activity of the yeast ribosome at the elongation step of translation.

The GGQ minidomain of the ribosomal protein eL42 was previously shown to contact the CCA-arm of P-site bound tRNA in human ribosome, indicating a possible involvement of the protein in the catalytic activity. Here, using Schizosaccharomyces pombe (S. pombe) cells, we demonstrate that the GGQ minidomain and neighboring region of eL42 is critical for the ribosomal function. Mutant eL42 proteins containing amino acid substitutions within or adjacent to the GGQ minidomain failed to complement the function of wild-type eL42, and expression of the mutant eL42 proteins led to severe growth defects. These results suggest that the mutations in eL42 interfere with the ribosomal function in vivo. Furthermore, we show that some of the mutations associated with the conserved GGQ region lead to reduced activities in the poly(Phe) synthesis and/or in the peptidyl transferase reaction with respect to puromycin, as compared with those of the wild-type ribosomes. A pK value of 6.95 was measured for the side chain of Lys-55/Arg-55, which is considerably less than that of a Lys or Arg residue. Altogether, our findings suggest that eL42 contributes to the 80S ribosome's peptidyl transferase activity by promoting the course of the elongation cycle.

Ribosome Structure, Function, and Early Evolution.

Ribosomes are among the largest and most dynamic molecular motors. The structure and dynamics of translation initiation and elongation are reviewed. Three ribosome motions have been identified for initiation and translocation. A swivel motion between the head/beak and the body of the 30S subunit was observed. A tilting dynamic of the head/beak versus the body of the 30S subunit was detected using simulations. A reversible ratcheting motion was seen between the 30S and the 50S subunits that slide relative to one another. The 30S⁻50S intersubunit contacts regulate translocation. IF2, EF-Tu, and EF-G are homologous G-protein GTPases that cycle on and off the same site on the ribosome. The ribosome, aminoacyl-tRNA synthetase (aaRS) enzymes, transfer ribonucleic acid (tRNA), and messenger ribonucleic acid (mRNA) form the core of information processing in cells and are coevolved. Surprisingly, class I and class II aaRS enzymes, with distinct and incompatible folds, are homologs. Divergence of class I and class II aaRS enzymes and coevolution of the genetic code are described by analysis of ancient archaeal species.

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.

Translational Control through Differential Ribosome Pausing during Amino Acid Limitation in Mammalian Cells.

Limitation for amino acids is thought to regulate translation in mammalian cells primarily by signaling through the kinases mTORC1 and GCN2. We find that a selective loss of arginine tRNA charging during limitation for arginine regulates translation through ribosome pausing at two of six arginine codons. Surprisingly, limitation for leucine, an essential and abundant amino acid in protein, results in little or no ribosome pausing. Chemical and genetic perturbation of mTORC1 and GCN2 signaling revealed that their robust response to leucine limitation prevents ribosome pausing, while an insufficient response to arginine limitation leads to loss of tRNA charging and ribosome pausing. Ribosome pausing decreases protein production and triggers premature ribosome termination without reducing mRNA levels. Together, our results suggest that amino acids that are not optimally sensed by the mTORC1 and GCN2 pathways still regulate translation through an evolutionarily conserved mechanism based on codon-specific ribosome pausing.

Translocation kinetics and structural dynamics of ribosomes are modulated by the conformational plasticity of downstream pseudoknots.

Downstream stable mRNA secondary structures can stall elongating ribosomes by impeding the concerted movements of tRNAs and mRNA on the ribosome during translocation. The addition of a downstream mRNA structure, such as a stem-loop or a pseudoknot, is essential to induce -1 programmed ribosomal frameshifting (-1 PRF). Interestingly, previous studies revealed that -1 PRF efficiencies correlate with conformational plasticity of pseudoknots, defined as their propensity to form incompletely folded structures, rather than with the mechanical properties of pseudoknots. To elucidate the detailed molecular mechanisms of translocation and -1 PRF, we applied several smFRET assays to systematically examine how translocation rates and conformational dynamics of ribosomes were affected by different pseudoknots. Our results show that initial pseudoknot-unwinding significantly inhibits late-stage translocation and modulates conformational dynamics of ribosomal post-translocation complexes. The effects of pseudoknots on the structural dynamics of ribosomes strongly correlate with their abilities to induce -1 PRF. Our results lead us to propose a kinetic scheme for translocation which includes an initial power-stroke step and a following thermal-ratcheting step. This scheme provides mechanistic insights on how selective modulation of late-stage translocation by pseudoknots affects -1 PRF. Overall our findings advance current understanding of translocation and ribosome-induced mRNA structure unwinding.

Mechanistic alternatives for peptide bond formation on the ribosome.

The peptidyl transfer reaction on the large ribosomal subunit depends on the protonation state of the amine nucleophile and exhibits a large kinetic solvent isotope effect (KSIE ∼8). In contrast, the related peptidyl-tRNA hydrolysis reaction involved in termination shows a KSIE of ∼4 and a pH-rate profile indicative of base catalysis. It is, however, unclear why these reactions should proceed with different mechanisms, as the experimental data suggests. One explanation is that two competing mechanisms may be operational in the peptidyl transferase center (PTC). Herein, we explored this possibility by re-examining the previously proposed proton shuttle mechanism and testing the feasibility of general base catalysis also for peptide bond formation. We employed a large cluster model of the active site and different reaction mechanisms were evaluated by density functional theory calculations. In these calculations, the proton shuttle and general base mechanisms both yield activation energies comparable to the experimental values. However, only the proton shuttle mechanism is found to be consistent with the experimentally observed pH-rate profile and the KSIE. This suggests that the PTC promotes the proton shuttle mechanism for peptide bond formation, while prohibiting general base catalysis, although the detailed mechanism by which general base catalysis is excluded remains unclear.

Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome.

During protein synthesis, a ribosome moves along the mRNA template and, using aminoacyl-tRNAs, decodes the template nucleotide triplets to assemble a protein amino acid sequence. This movement is accompanied by shifting of mRNA-tRNA complexes within the ribosome in a process called translocation. In living cells, this process proceeds in a unidirectional manner, bringing the ribosome to the 3' end of mRNA, and is catalyzed by the GTPase translation elongation factor 2 (EF-G in prokaryotes and eEF2 in eukaryotes). Interestingly, the possibility of spontaneous backward translocation has been shown in vitro for bacterial ribosomes, suggesting a potential reversibility of this reaction. However, this possibility has not yet been tested for eukaryotic ribosomes. Here, using a reconstituted mammalian translation system, we show that the eukaryotic elongation factor eEF2 catalyzes ribosomal reverse translocation at one mRNA triplet. We found that this process requires a cognate tRNA in the ribosomal E-site and cannot occur spontaneously without eEF2. The efficiency of this reaction depended on the concentrations of eEF2 and cognate tRNAs and increased in the presence of nonhydrolyzable GTP analogues. Of note, ADP-ribosylation of eEF2 domain IV blocked reverse translocation, suggesting a crucial role of interactions of this domain with the ribosome for the catalysis of the reaction. In summary, our findings indicate that eEF2 is able to induce ribosomal translocation in forward and backward directions, highlighting the universal mechanism of tRNA-mRNA movements within the ribosome.