Emerging Personalized Opportunities for Enhancing Translational Readthrough in Rare Genetic Diseases and Beyond.

Nonsense mutations trigger premature translation termination and often give rise to prevalent and rare genetic diseases. Consequently, the pharmacological suppression of an unscheduled stop codon represents an attractive treatment option and is of high clinical relevance. At the molecular level, the ability of the ribosome to continue translation past a stop codon is designated stop codon readthrough (SCR). SCR of disease-causing premature termination codons (PTCs) is minimal but small molecule interventions, such as treatment with aminoglycoside antibiotics, can enhance its frequency. In this review, we summarize the current understanding of translation termination (both at PTCs and at cognate stop codons) and highlight recently discovered pathways that influence its fidelity. We describe the mechanisms involved in the recognition and readthrough of PTCs and report on SCR-inducing compounds currently explored in preclinical research and clinical trials. We conclude by reviewing the ongoing attempts of personalized nonsense suppression therapy in different disease contexts, including the genetic skin condition epidermolysis bullosa.

Visualizing translation dynamics at atomic detail inside a bacterial cell.

Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells1. Here we use advances in cryo-electron tomography and sub-tomogram analysis2,3 to visualize the structural dynamics of translation inside the bacterium Mycoplasma pneumoniae. To interpret the functional states in detail, we first obtain a high-resolution in-cell average map of all translating ribosomes and build an atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves 13 ribosome states that differ in their conformation and composition. These recapitulate major states that were previously resolved in vitro, and reflect intermediates during active translation. On the basis of these states, we animate translation elongation inside native cells and show how antibiotics reshape the cellular translation landscapes. During translation elongation, ribosomes often assemble in defined three-dimensional arrangements to form polysomes4. By mapping the intracellular organization of translating ribosomes, we show that their association into polysomes involves a local coordination mechanism that is mediated by the ribosomal protein L9. We propose that an extended conformation of L9 within polysomes mitigates collisions to facilitate translation fidelity. Our work thus demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells.

Discovery of C13-Aminobenzoyl Cycloheximide Derivatives that Potently Inhibit Translation Elongation.

Employed for over half a century to study protein synthesis, cycloheximide (CHX, 1) is a small molecule natural product that reversibly inhibits translation elongation. More recently, CHX has been applied to ribosome profiling, a method for mapping ribosome positions on mRNA genome-wide. Despite CHX's extensive use, CHX treatment often results in incomplete translation inhibition due to its rapid reversibility, prompting the need for improved reagents. Here, we report the concise synthesis of C13-amide-functionalized CHX derivatives with increased potencies toward protein synthesis inhibition. Cryogenic electron microscopy (cryo-EM) revealed that C13-aminobenzoyl CHX (8) occupies the same site as CHX, competing with the 3' end of E-site tRNA. We demonstrate that 8 is superior to CHX for ribosome profiling experiments, enabling more effective capture of ribosome conformations through sustained stabilization of polysomes. Our studies identify powerful chemical reagents to study protein synthesis and reveal the molecular basis of their enhanced potency.

The porphyrin TMPyP4 inhibits elongation during the noncanonical translation of the FTLD/ALS-associated GGGGCC repeat in the C9orf72 gene.

GGGGCC (G4C2) repeat expansion in the C9orf72 gene has been shown to cause frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Dipeptide repeat proteins produced through repeat-associated non-AUG (RAN) translation are recognized as potential drivers for neurodegeneration. Therefore, selective inhibition of RAN translation could be a therapeutic avenue to treat these neurodegenerative diseases. It was previously known that the porphyrin TMPyP4 binds to G4C2 repeat RNA. However, the consequences of this interaction have not been well characterized. Here, we confirmed that TMPyP4 inhibits C9orf72 G4C2 repeat translation in cellular and in in vitro translation systems. An artificial insertion of an AUG codon failed to cancel the translation inhibition, suggesting that TMPyP4 acts downstream of non-AUG translation initiation. Polysome profiling assays also revealed polysome retention on G4C2 repeat RNA, along with inhibition of translation, indicating that elongating ribosomes stall on G4C2 repeat RNA. Urea-resistant interaction between G4C2 repeat RNA and TMPyP4 likely contributes to this ribosome stalling and thus to selective inhibition of RAN translation. Taken together, our data reveal a novel mode of action of TMPyP4 as an inhibitor of G4C2 repeat translation elongation.

Blasticidin S inhibits mammalian translation and enhances production of protein encoded by nonsense mRNA.

Deciphering translation is of paramount importance for the understanding of many diseases, and antibiotics played a pivotal role in this endeavour. Blasticidin S (BlaS) targets translation by binding to the peptidyl transferase center of the large ribosomal subunit. Using biochemical, structural and cellular approaches, we show here that BlaS inhibits both translation elongation and termination in Mammalia. Bound to mammalian terminating ribosomes, BlaS distorts the 3'CCA tail of the P-site tRNA to a larger extent than previously reported for bacterial ribosomes, thus delaying both, peptide bond formation and peptidyl-tRNA hydrolysis. While BlaS does not inhibit stop codon recognition by the eukaryotic release factor 1 (eRF1), it interferes with eRF1's accommodation into the peptidyl transferase center and subsequent peptide release. In human cells, BlaS inhibits nonsense-mediated mRNA decay and, at subinhibitory concentrations, modulates translation dynamics at premature termination codons leading to enhanced protein production.

Ataluren and aminoglycosides stimulate read-through of nonsense codons by orthogonal mechanisms.

During protein synthesis, nonsense mutations, resulting in premature stop codons (PSCs), produce truncated, inactive protein products. Such defective gene products give rise to many diseases, including cystic fibrosis, Duchenne muscular dystrophy (DMD), and some cancers. Small molecule nonsense suppressors, known as TRIDs (translational read-through-inducing drugs), stimulate stop codon read-through. The best characterized TRIDs are ataluren, which has been approved by the European Medicines Agency for the treatment of DMD, and G418, a structurally dissimilar aminoglycoside. Previously [1], we applied a highly purified in vitro eukaryotic translation system to demonstrate that both aminoglycosides like G418 and more hydrophobic molecules like ataluren stimulate read-through by direct interaction with the cell's protein synthesis machinery. Our results suggested that they might do so by different mechanisms. Here, we pursue this suggestion through a more-detailed investigation of ataluren and G418 effects on read-through. We find that ataluren stimulation of read-through derives exclusively from its ability to inhibit release factor activity. In contrast, G418 increases functional near-cognate tRNA mispairing with a PSC, resulting from binding to its tight site on the ribosome, with little if any effect on release factor activity. The low toxicity of ataluren suggests that development of new TRIDs exclusively directed toward inhibiting termination should be a priority in combatting PSC diseases. Our results also provide rate measurements of some of the elementary steps during the eukaryotic translation elongation cycle, allowing us to determine how these rates are modified when cognate tRNA is replaced by near-cognate tRNA ± TRIDs.

Translation elongation rate varies among organs and decreases with age.

There has been a surge of interest towards targeting protein synthesis to treat diseases and extend lifespan. Despite the progress, few options are available to assess translation in live animals, as their complexity limits the repertoire of experimental tools to monitor and manipulate processes within organs and individual cells. It this study, we developed a labeling-free method for measuring organ- and cell-type-specific translation elongation rates in vivo. It is based on time-resolved delivery of translation initiation and elongation inhibitors in live animals followed by ribosome profiling. It also reports translation initiation sites in an organ-specific manner. Using this method, we found that the elongation rates differ more than 50% among mouse organs and determined them to be 6.8, 5.0 and 4.3 amino acids per second for liver, kidney, and skeletal muscle, respectively. We further found that the elongation rate is reduced by 20% between young adulthood and mid-life. Thus, translation, a major metabolic process in cells, is tightly regulated at the level of elongation of nascent polypeptide chains.

Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes.

Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti-puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides in mammalian cells, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise subcellular translation sites using anti-puromycin immunostaining may be confounded by release of puromycylated nascent polypeptide chains prior to fixation.

Nutrient-dependent control of RNA polymerase II elongation rate regulates specific gene expression programs by alternative polyadenylation.

Transcription by RNA polymerase II (RNAPII) is a dynamic process with frequent variations in the elongation rate. However, the physiological relevance of variations in RNAPII elongation kinetics has remained unclear. Here we show in yeast that a RNAPII mutant that reduces the transcription elongation rate causes widespread changes in alternative polyadenylation (APA). We unveil two mechanisms by which APA affects gene expression in the slow mutant: 3' UTR shortening and gene derepression by premature transcription termination of upstream interfering noncoding RNAs. Strikingly, the genes affected by these mechanisms are enriched for functions involved in phosphate uptake and purine synthesis, processes essential for maintenance of the intracellular nucleotide pool. As nucleotide concentration regulates transcription elongation, our findings argue that RNAPII is a sensor of nucleotide availability and that genes important for nucleotide pool maintenance have adopted regulatory mechanisms responsive to reduced rates of transcription elongation.

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.

Ribosome profiling in archaea reveals leaderless translation, novel translational initiation sites, and ribosome pausing at single codon resolution.

High-throughput methods, such as ribosome profiling, have revealed the complexity of translation regulation in Bacteria and Eukarya with large-scale effects on cellular functions. In contrast, the translational landscape in Archaea remains mostly unexplored. Here, we developed ribosome profiling in a model archaeon, Haloferax volcanii, elucidating, for the first time, the translational landscape of a representative of the third domain of life. We determined the ribosome footprint of H. volcanii to be comparable in size to that of the Eukarya. We linked footprint lengths to initiating and elongating states of the ribosome on leadered transcripts, operons, and on leaderless transcripts, the latter representing 70% of H. volcanii transcriptome. We manipulated ribosome activity with translation inhibitors to reveal ribosome pausing at specific codons. Lastly, we found that the drug harringtonine arrested ribosomes at initiation sites in this archaeon. This drug treatment allowed us to confirm known translation initiation sites and also reveal putative novel initiation sites in intergenic regions and within genes. Ribosome profiling revealed an uncharacterized complexity of translation in this archaeon with bacteria-like, eukarya-like, and potentially novel translation mechanisms. These mechanisms are likely to be functionally essential and to contribute to an expanded proteome with regulatory roles in gene expression.

N6-Methyladenosine Regulates the Expression and Secretion of TGFß1 to Affect the Epithelial-Mesenchymal Transition of Cancer Cells.

N6-methyladenosine (m6A) is the most abundant modification on eukaryotic mRNA, which regulates all steps of the mRNA life cycle. An increasing number of studies have shown that m6A methylation plays essential roles in tumor development. However, the relationship between m6A and the progression of cancers remains to be explored. Here, we reported that transforming growth factor-ß (TGFß1)-induced epithelial-mesenchymal transition (EMT) was inhibited in methyltransferase-like 3 (METTL3) knockdown (Mettl3Mut/-) cells. The expression of TGFß1 was up-regulated, while self-stimulated expression of TGFß1 was suppressed in Mettl3Mut/- cells. We further revealed that m6A promoted TGFB1 mRNA decay, but impaired TGFB1 translation progress. Besides this, the autocrine of TGFß1 was disrupted in Mettl3Mut/- cells via interrupting TGFß1 dimer formation. Lastly, we found that Snail, which was down-regulated in Mettl3Mut/- cells, was a key factor responding to TGFß1-induced EMT. Together, our research demonstrated that m6A performed multi-functional roles in TGFß1 expression and EMT modulation, suggesting the critical roles of m6A in cancer progression regulation.

Inhibiting Translation Elongation with SVC112 Suppresses Cancer Stem Cells and Inhibits Growth in Head and Neck Squamous Carcinoma.

Cancer stem cells (CSC) drive growth, therapy resistance, and recurrence in head and neck squamous cell carcinoma (HNSCC). Regulation of protein translation is crucial for normal stem cells and CSCs; its inhibition could disrupt stemness properties, but translation inhibitors are limited clinically due to toxicity. SVC112 is a synthetic derivative of bouvardin, a plant-derived translation elongation inhibitor. SVC112 had greater antiproliferative effects on HNSCC cells compared with the FDA-approved translation inhibitor omacetaxine mepesuccinate (HHT). SVC112 preferentially inhibited cancer cells compared with patient-matched cancer-associated fibroblasts, whereas HHT was equally toxic to both. SVC112 reduced sphere formation by cell lines and CSCs. SVC112 alone inhibited the growth of patient-derived xenografts (PDX), and SVC112 combined with radiation resulted in tumor regression in HPV-positive and HPV-negative HNSCC PDXs. Notably, CSC depletion after SVC112 correlated with tumor response. SVC112 preferentially impeded ribosomal processing of mRNAs critical for stress response and decreased CSC-related proteins including Myc and Sox2. SVC112 increased cell-cycle progression delay and slowed DNA repair following radiation, enhancing colony and sphere formation radiation effects. In summary, these data demonstrate that SVC112 suppresses CSC-related proteins, enhances the effects of radiation, and blocks growth of HNSCC PDXs by inhibiting CSCs. SIGNIFICANCE: Inhibiting protein elongation with SVC112 reduces tumor growth in head and neck squamous cell carcinoma and increases the effects of radiation by targeting the cancer stem cell pool.

Synthetic Antibody Binding to a Preorganized RNA Domain of Hepatitis C Virus Internal Ribosome Entry Site Inhibits Translation.

Structured RNA elements within the internal ribosome entry site (IRES) of hepatitis C virus (HCV) genome hijack host cell machinery for translation initiation through a cap-independent mechanism. Here, using a phage display selection, we obtained two antibody fragments (Fabs), HCV2 and HCV3, against HCV IRES that bind the RNA with dissociation constants of 32 ± 7 nM and 37 ± 8 nM respectively, specifically recognizing the so-called junction IIIabc (JIIIabc). We used these Fabs as crystallization chaperones and determined the high-resolution crystal structures of JIIIabc-HCV2 and -HCV3 complexes at 1.81 Å and 2.75 Å resolution respectively, revealing an antiparallel four-way junction with the IIIa and IIIc subdomains brought together through tertiary interactions. The RNA conformation observed in the structures supports the structural model for this region derived from cryo-EM data for the HCV IRES-40S ribosome complex, suggesting that the tertiary fold of the RNA preorganizes the domain for interactions with the 40S ribosome. Strikingly, both Fabs and the ribosomal protein eS27 not only interact with a common subset of nucleotides within the JIIIabc but also use physiochemically similar sets of protein residues to do so, suggesting that the RNA surface is well-suited for interactions with proteins, perhaps analogous to the "hot spot" concept elaborated for protein-protein interactions. Using a rabbit reticulocyte lysate-based translation assay with a bicistronic reporter construct, we further demonstrated that Fabs HCV2 and HCV3 specifically inhibit the HCV IRES-directed translation, implicating disruption of the JIIIabc-ribosome interaction as a potential therapeutic strategy against HCV.

The hidden nature of protein translational control by diphthamide: the secrets under the leather.

The protein translation elongation factor eEF2 undergoes a unique posttranslational modification called diphthamidation. eEF2 is an essential factor in protein translation, and the diphthamide modification has been a famous target of the diphtheria toxin for a long time. On the other hand, the physiological function of this rare modification in vivo remains unknown. Recent studies have suggested that diphthamide has specific functions for the cellular stress response and active proliferation. In this review, we summarize the history and findings of diphthamide obtained to date and discuss the possibility of a specific function for diphthamide in regulating protein translation.

How Messenger RNA and Nascent Chain Sequences Regulate Translation Elongation.

Translation elongation is a highly coordinated, multistep, multifactor process that ensures accurate and efficient addition of amino acids to a growing nascent-peptide chain encoded in the sequence of translated messenger RNA (mRNA). Although translation elongation is heavily regulated by external factors, there is clear evidence that mRNA and nascent-peptide sequences control elongation dynamics, determining both the sequence and structure of synthesized proteins. Advances in methods have driven experiments that revealed the basic mechanisms of elongation as well as the mechanisms of regulation by mRNA and nascent-peptide sequences. In this review, we highlight how mRNA and nascent-peptide elements manipulate the translation machinery to alter the dynamics and pathway of elongation.

Genome-wide Translation Profiling by Ribosome-Bound tRNA Capture.

In the ribosome complex, tRNA is a critical element of mRNA translation. A rich repertoire of cell regulation is hypothesized to occur during the recruitment of specific tRNAs in polypeptide formation. However, this basic question in nascent chain biology remains unaddressed due to the lack of technologies to report the complete tRNA complement inside ribosomes during active translation. Here, we characterize a technique for profiling ribosome-embedded tRNA and their modifications. With this method, we generated a comprehensive survey of the quantity and quality of intra-ribosomal tRNAs. In cells under environmental stress, we show that methionine tRNA inside ribosomes is a robust biomarker for the impairment of translation initiation or elongation steps. Concurrent tRNA/mRNA ribosome profiling revealed a stress-dependent incorporation of damaged and uncharged tRNAs into ribosomes causing translation arrest. Thus, tRNA ribosome profiling can provide insights on translation control mechanisms in diverse biological contexts.

Effect of Fusidic Acid on the Kinetics of Molecular Motions During EF-G-Induced Translocation on the Ribosome.

The translocation step of protein synthesis entails binding and dissociation of elongation factor G (EF-G), movements of the two tRNA molecules, and motions of the ribosomal subunits. The translocation step is targeted by many antibiotics. Fusidic acid (FA), an antibiotic that blocks EF-G on the ribosome, may also interfere with some of the ribosome rearrangements, but the exact timing of inhibition remains unclear. To follow in real-time the dynamics of the ribosome-tRNA-EF-G complex, we have developed a fluorescence toolbox which allows us to monitor the key molecular motions during translocation. Here we employed six different fluorescence observables to investigate how FA affects translocation kinetics. We found that FA binds to an early translocation intermediate, but its kinetic effect on tRNA movement is small. FA does not affect the synchronous forward (counterclockwise) movements of the head and body domains of the small ribosomal subunit, but exerts a strong effect on the rates of late translocation events, i.e. backward (clockwise) swiveling of the head domain and the transit of deacylated tRNA through the E' site, in addition to blocking EF-G dissociation. The use of ensemble kinetics and numerical integration unraveled how the antibiotic targets molecular motions within the ribosome-EF-G complex.

Comprehensive characterization of a theophylline riboswitch reveals two pivotal features of Shine-Dalgarno influencing activated translation property.

Tuneable gene expression controlled by synthetic biological elements is of great importance to biotechnology and synthetic biology. The synthetic riboswitch is a pivotal type of elements that can easily control the heterologous gene expression in diverse bacteria. In this study, the theophylline-dependent synthetic riboswitch and the corresponding variants with varied spacings between Shine-Dalgarno (SD) sequence and start codon were employed to comprehensively characterize the induction and regulation properties through combining a strong promoter aprE in Bacillus subtilis. Amongst the sets of newly constructed expression elements, the expression element with 9-bp spacing exhibited the higher expression level, a superior induction fold performance, and a considerably lower leaky expression than those with longer or shorter spacings. The riboswitch expression element with 9-bp spacing showed an approximately linear dose dependence from 0 to 8 mM of theophylline. Modification of the SD sequence through the insertion of a single A base prior to the native sequence enables the increase of the expression level post induction while decreasing the induction fold as a result of the elevated leaky level. The riboswitch elements with the engineered SD and the optimal 9-bp spacing exhibit an altered dose dependency in which the approximately linear range shifts to 0-4 mM, although it has a similar profile to the induction process. These results not only provide comprehensive data for the induced expression by a theophylline riboswitch combined with a strong native promoter from B. subtilis but also provide the two pivotal features of SD essential to the modular design of other synthetic riboswitches.