Translation Elongation Rate Research Articles

Human DDX6 regulates translation and decay of inefficiently translated mRNAs.

Recent findings indicate that the translation elongation rate influences mRNA stability. One of the factors that has been implicated in this link between mRNA decay and translation speed is the yeast DEAD-box helicase Dhh1p. Here, we demonstrated that the human ortholog of Dhh1p, DDX6, triggers the deadenylation-dependent decay of inefficiently translated mRNAs in human cells. DDX6 interacts with the ribosome through the Phe-Asp-Phe (FDF) motif in its RecA2 domain. Furthermore, RecA2-mediated interactions and ATPase activity are both required for DDX6 to destabilize inefficiently translated mRNAs. Using ribosome profiling and RNA sequencing, we identified two classes of endogenous mRNAs that are regulated in a DDX6-dependent manner. The identified targets are either translationally regulated or regulated at the steady-state-level and either exhibit signatures of poor overall translation or of locally reduced ribosome translocation rates. Transferring the identified sequence stretches into a reporter mRNA caused translation- and DDX6-dependent degradation of the reporter mRNA. In summary, these results identify DDX6 as a crucial regulator of mRNA translation and decay triggered by slow ribosome movement and provide insights into the mechanism by which DDX6 destabilizes inefficiently translated mRNAs.

Growth-rate dependency of ribosome abundance and translation elongation rate in Corynebacterium glutamicum differs from that in Escherichia coli

Bacterial growth rate (µ) depends on the protein synthesis capacity of the cell and thus on the number of active ribosomes and their translation elongation rate. The relationship between these fundamental growth parameters have only been described for few bacterial species, in particular Escherichia coli. Here, we analyse the growth-rate dependency of ribosome abundance and translation elongation rate for Corynebacterium glutamicum, a gram-positive model species differing from E. coli by a lower growth temperature optimum and a lower maximal growth rate. We show that, unlike in E. coli, there is little change in ribosome abundance for µ <0.4 h−1 in C. glutamicum and the fraction of active ribosomes is kept above 70% while the translation elongation rate declines 5-fold. Mathematical modelling indicates that the decrease in the translation elongation rate can be explained by a depletion of translation precursors.

Single-molecule imaging reveals distinct elongation and frameshifting dynamics between frames of expanded RNA repeats in C9ORF72-ALS/FTD

C9ORF72 hexanucleotide repeat expansion is the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One pathogenic mechanism is the accumulation of toxic dipeptide repeat (DPR) proteins like poly-GA, GP and GR, produced by the noncanonical translation of the expanded RNA repeats. However, how different DPRs are synthesized remains elusive. Here, we use single-molecule imaging techniques to directly measure the translation dynamics of different DPRs. Besides initiation, translation elongation rates vary drastically between different frames, with GP slower than GA and GR the slowest. We directly visualize frameshift events using a two-color single-molecule translation assay. The repeat expansion enhances frameshifting, but the overall frequency is low. There is a higher chance of GR-to-GA shift than in the reversed direction. Finally, the ribosome-associated protein quality control (RQC) factors ZNF598 and Pelota modulate the translation dynamics, and the repeat RNA sequence is important for invoking the RQC pathway. This study reveals that multiple translation steps modulate the final DPR production. Understanding repeat RNA translation is critically important to decipher the DPR-mediated pathogenesis and identify potential therapeutic targets in C9ORF72-ALS/FTD.

MRNA location and translation rate determine protein targeting to dual destinations

Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that mRNA location and translation rate can also determine protein targeting by modulating protein binding to specific interacting partners. Peripheral localization of the NET1 mRNA and fast translation lead to higher cytosolic retention of the NET1 protein by promoting its binding to the membrane-associated scaffold protein CASK. By contrast, perinuclear mRNA location and/or slower translation rate favor nuclear targeting by promoting binding to importins. This mRNA location-dependent mechanism is modulated by physiological stimuli and profoundly impacts NET1 function in cell motility. These results reveal that the location of protein synthesis and the rate of translation elongation act in coordination as a "partner-selection" mechanism that robustly influences protein distribution and function.

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.

In Situ Self‐Assembly of Fluorogenic RNA Nanozipper Enables Real‐Time Imaging of Single Viral mRNA Translation

AbstractReal‐time visualization of individual viral mRNA translation activities in live cells is essential to obtain critical details of viral mRNA dynamics and to detect its transient responses to environmental stress. Fluorogenic RNA aptamers are powerful tools for real‐time imaging of mRNA in live cells, but monitoring the translation activity of individual mRNAs remains a challenge due to their intrinsic photophysical properties. Here, we develop a genetically encoded turn‐on 3,5‐difluoro‐4‐hydroxybenzylidene imidazolinone (DFHBI)‐binding RNA nanozipper with superior brightness and high photostability by in situ self‐assembly of multiple nanozippers along single mRNAs. The nanozipper enables real‐time imaging of the mobility and dynamic translation of individual viral mRNAs in live cells, providing information on the spatial dynamics and translational elongation rate of viral mRNAs.

In Situ Self-Assembly of Fluorogenic RNA Nanozipper Enables Real-Time Imaging of Single Viral mRNA Translation.

Real-time visualization of individual viral mRNA translation activities in live cells is essential to obtain critical details of viral mRNA dynamics and to detect its transient responses to environmental stress. Fluorogenic RNA aptamers are powerful tools for real-time imaging of mRNA in live cells, but monitoring the translation activity of individual mRNAs remains a challenge due to their intrinsic photophysical properties. Here, we develop a genetically encoded turn-on 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI)-binding RNA nanozipper with superior brightness and high photostability by in situ self-assembly of multiple nanozippers along single mRNAs. The nanozipper enables real-time imaging of the mobility and dynamic translation of individual viral mRNAs in live cells, providing information on the spatial dynamics and translational elongation rate of viral mRNAs.

Translation and mRNA Stability Control.

Messenger RNA (mRNA) stability and translational efficiency are two crucial aspects of the post-transcriptional process that profoundly impact protein production in a cell. While it is widely known that ribosomes produce proteins, studies during the past decade have surprisingly revealed that ribosomes also control mRNA stability in a codon-dependent manner, a process referred to as codon optimality. Therefore, codons, the three-nucleotide words read by the ribosome, have a potent effect on mRNA stability and provide cis-regulatory information that extends beyond the amino acids they encode. While the codon optimality molecular mechanism is still unclear, the translation elongation rate appears to trigger mRNA decay. Thus, transfer RNAs emerge as potential master gene regulators affecting mRNA stability. Furthermore, while few factors related to codon optimality have been identified in yeast, the orthologous genes in vertebrates do not necessary share the same functions. Here, we discuss codon optimality findings and gene regulation layers related to codon composition in different eukaryotic species.

Revisiting the growth rate hypothesis: Towards a holistic stoichiometric understanding of growth.

The growth rate hypothesis (GRH) posits that variation in organismal stoichiometry (C:P and N:P ratios) is driven by growth-dependent allocation of P to ribosomal RNA. The GRH has found broad but not uniform support in studies across diverse biota and habitats. We synthesise information on how and why the tripartite growth-RNA-P relationship predicted by the GRH may be uncoupled and outline paths for both theoretical and empirical work needed to broaden the working domain of the GRH. We found strong support for growth to RNA (r2 = 0.59) and RNA-P to P (r2 = 0.63) relationships across taxa, but growth to P relationships were relatively weaker (r2 = 0.09). Together, the GRH was supported in ~50% of studies. Mechanisms behind GRH uncoupling were diverse but could generally be attributed to physiological (P accumulation in non-RNA pools, inactive ribosomes, translation elongation rates and protein turnover rates), ecological (limitation by resources other than P), and evolutionary (adaptation to different nutrient supply regimes) causes. These factors should be accounted for in empirical tests of the GRH and formalised mathematically to facilitate a predictive understanding of growth.

Maintenance of tRNA and elongation factors supports T3SS proteins translational elongations in pathogenic bacteria during nutrient starvation

BackgroundSufficient nutrition contributes to rapid translational elongation and protein synthesis in eukaryotic cells and prokaryotic bacteria. Fast synthesis and accumulation of type III secretion system (T3SS) proteins conduce to the invasion of pathogenic bacteria into the host cells. However, the translational elongation patterns of T3SS proteins in pathogenic bacteria under T3SS-inducing conditions remain unclear. Here, we report a mechanism of translational elongation of T3SS regulators, effectors and structural protein in four model pathogenic bacteria (Pseudomonas syringae, Pseudomonas aeruginosa, Xanthomonas oryzae and Ralstonia solanacearum) and a clinical isolate (Pseudomonas aeruginosa UCBPP-PA14) under nutrient-limiting conditions. We proposed a luminescence reporter system to quantitatively determine the translational elongation rates (ERs) of T3SS regulators, effectors and structural protein under different nutrient-limiting conditions and culture durations.ResultsThe translational ERs of T3SS regulators, effectors and structural protein in these pathogenic bacteria were negatively regulated by the nutrient concentration and culture duration. The translational ERs in 0.5× T3SS-inducing medium were the highest of all tested media. In 1× T3SS-inducing medium, the translational ERs were highest at 0 min and then rapidly decreased. The translational ERs of T3SS regulators, effectors and structural protein were inhibited by tRNA degradation and by reduced levels of elongation factors (EFs).ConclusionsRapid translational ER and synthesis of T3SS protein need adequate tRNAs and EFs in nutrient-limiting conditions. Numeric presentation of T3SS translation visually indicates the invasion of bacteria and provides new insights into T3SS expression that can be applied to other pathogenic bacteria.

Context‐specific roles of diphthamide deficiency in hepatocellular carcinogenesis

Diphthamide biosynthesis protein 1 (DPH1) is biochemically involved in the first step of diphthamide biosynthesis, a post-translational modification of eukaryotic elongation factor 2 (EEF2). Earlier studies showed that DPH1, also known as ovarian cancer-associated gene 1 (OVCA1), is involved in ovarian carcinogenesis. However, the role of DPH1 in hepatocellular carcinoma (HCC) remains unclear. To investigate the impact of DPH1 in hepatocellular carcinogenesis, we performed data mining from The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) dataset. We found that reduced DPH1 levels were associated with advanced stages and poor survival of patients with HCC. Also, we generated hepatocyte-specific Dph1-deficient mice and showed that diphthamide-deficient EEF2 resulted in a reduced translation elongation rate in the hepatocytes and led to mild liver damage with fatty accumulation. After N-diethylnitrosamine (DEN)-induced acute liver injury, p53-mediated pericentral hepatocyte death was increased, and compensatory proliferation was reduced in Dph1-deficient mice. Consistent with these effects, Dph1 deficiency decreased the incidence of DEN-induced pericentral-derived HCC and revealed a protective effect against p53 loss. In contrast, Dph1 deficiency combined with Trp53- or Trp53/Pten-deficient hepatocytes led to increased tumor loads associated with KRT19 (K19)-positive periportal-like cell expansion in mice. Further gene set enrichment analysis also revealed that HCC patients with lower levels of DPH1 and TP53 expression had enriched gene-sets related to the cell cycle and K19-upregulated HCC. Additionally, liver tumor organoids obtained from 6-month-old Pten/Trp53/Dph1-triple-mutant mice had a higher frequency of organoid re-initiation cells and higher proliferative index compared with those of the Pten/Trp53-double-mutant. Pten/Trp53/Dph1-triple-mutant liver tumor organoids showed expression of genes associated with stem/progenitor phenotypes, including Krt19 and Prominin-1 (Cd133) progenitor markers, combined with low hepatocyte-expressed fibrinogen genes. These findings indicate that diphthamide deficiency differentially regulates hepatocellular carcinogenesis, which inhibits pericentral hepatocyte-derived tumors and promotes periportal progenitor-associated liver tumors. © 2022 The Pathological Society of Great Britain and Ireland.

Chaperone requirements for de novo folding of Saccharomyces cerevisiae septins.

Polymers of septin protein complexes play cytoskeletal roles in eukaryotic cells. The specific subunit composition within complexes controls functions and higher-order structural properties. All septins have globular GTPase domains. The other eukaryotic cytoskeletal NTPases strictly require assistance from molecular chaperones of the cytosol, particularly the cage-like chaperonins, to fold into oligomerization-competent conformations. We previously identified cytosolic chaperones that bind septins and influence the oligomerization ability of septins carrying mutations linked to human disease, but it was unknown to what extent wild-type septins require chaperone assistance for their native folding. Here we use a combination of in vivo and in vitro approaches to demonstrate chaperone requirements for de novo folding and complex assembly by budding yeast septins. Individually purified septins adopted nonnative conformations and formed nonnative homodimers. In chaperonin- or Hsp70-deficient cells, septins folded slower and were unable to assemble posttranslationally into native complexes. One septin, Cdc12, was so dependent on cotranslational chaperonin assistance that translation failed without it. Our findings point to distinct translation elongation rates for different septins as a possible mechanism to direct a stepwise, cotranslational assembly pathway in which general cytosolic chaperones act as key intermediaries.

Ribo-DT: An automated pipeline for inferring codon dwell times from ribosome profiling data.

Protein synthesis is an energy consuming process characterised as a pivotal and highly regulated step in gene expression. The net protein output is dictated by a combination of translation initiation, elongation and termination rates that have remained difficult to measure. Recently, the development of ribosome profiling has enabled the inference of translation parameters through modelling, as this method informs on the ribosome position along the mRNA. Here, we present an automated, reproducible and portable computational pipeline to infer relative single-codon and codon-pair dwell times as well as gene flux from raw ribosome profiling sequencing data. As a case study, we applied our workflow to a publicly available yeast ribosome profiling dataset consisting of 57 independent gene knockouts related to RNA and tRNA modifications. We uncovered the effects of those modifications on translation elongation and codon selection during decoding. In particular, knocking out mod5 and trm7 increases codon-specific dwell times which indicates their potential tRNA targets, and highlights effects of nucleotide modifications on ribosome decoding rate.

FMRP-dependent production of large dosage-sensitive proteins is highly conserved.

Mutations in FMR1 are the most common heritable cause of autism spectrum disorder. FMR1 encodes an RNA-binding protein, FMRP, which binds to long, autism-relevant transcripts and is essential for normal neuronal and ovarian development. In contrast to the prevailing model that FMRP acts to block translation elongation, we previously found that FMRP activates the translation initiation of large proteins in Drosophila oocytes. We now provide evidence that FMRP-dependent translation is conserved and occurs in the mammalian brain. Our comparisons of the mammalian cortex and Drosophila oocyte ribosome profiling data show that translation of FMRP-bound mRNAs decreases to a similar magnitude in FMRP-deficient tissues from both species. The steady-state levels of several FMRP targets were reduced in the Fmr1 KO mouse cortex, including a ∼50% reduction of Auts2, a gene implicated in an autosomal dominant autism spectrum disorder. To distinguish between effects on elongation and initiation, we used a novel metric to detect the rate-limiting ribosome stalling. We found no evidence that FMRP target protein production is governed by translation elongation rates. FMRP translational activation of large proteins may be critical for normal human development, as more than 20 FMRP targets including Auts2 are dosage sensitive and are associated with neurodevelopmental disorders caused by haploinsufficiency.

Codon pair optimization (CPO): a software tool for synthetic gene design based on codon pair bias to improve the expression of recombinant proteins in Pichia pastoris

BackgroundCodon optimization is a common method to improve protein expression levels in Pichia pastoris and the current strategy is to replace rare codons with preferred codons to match the codon usage bias. However, codon-pair contexts have a profound effect on translation efficiency by influencing both translational elongation rates and accuracy. Until now, it remains untested whether optimized genes based on codon pair bias results in higher protein expression levels compared to codon usage bias.ResultsIn this study, an algorithm based on dynamic programming was introduced to develop codon pair optimization (CPO) which is a software tool to provide simple and efficient codon pair optimization for synthetic gene design in Pichia pastoris. Two reporters (MT1-MMP E2C6 and ADAM17 A9B8 scFvs) were employed to test the effects of codon pair bias and CPO optimization on their protein expression levels. Four variants of MT1-MMP E2C6 and ADAM17 A9B8 for each were generated, one variant with the best codon-pair context, one with the worst codon-pair context, one with unbiased codon-pair context, and another optimized based on codon usage. The expression levels of variants with the worst codon-pair context were almost undetectable by Western blot and the variants with the best codon-pair context were expressed well. The expression levels on MT1-MMP E2C6 and ADAM17 A9B8 were more than five times and seven times higher in the optimized sequences based on codon-pair context compared to that based on codon usage, respectively. The results indicated that the codon-pair context-based codon optimization is more effective in enhancing expression of protein in Pichia pastoris.ConclusionsCodon-pair context plays an important role on the protein expression in Pichia pastoris. The codon pair optimization (CPO) software developed in this study efficiently improved the protein expression levels of exogenous genes in Pichia pastoris, suggesting gene design based on codon pair bias is an alternative strategy for high expression of recombinant proteins in Pichia pastoris.

Roles of mRNA poly(A) tails in regulation of eukaryotic gene expression.

In eukaryotes, poly(A) tails are present on almost every mRNA. Early experiments led to the hypothesis that poly(A) tails and the cytoplasmic polyadenylate-binding protein (PABPC) promote translation and prevent mRNA degradation, but the details remained unclear. More recent data suggest that the role of poly(A) tails is much more complex: poly(A)-binding protein can stimulate poly(A) tail removal (deadenylation) and the poly(A) tails of stable, highly translated mRNAs at steady state are much shorter than expected. Furthermore, the rate of translation elongation affects deadenylation. Consequently, the interplay between poly(A) tails, PABPC, translation and mRNA decay has a major role in gene regulation. In this Review, we discuss recent work that is revolutionizing our understanding of the roles of poly(A) tails in the cytoplasm. Specifically, we discuss the roles of poly(A) tails in translation and control of mRNA stability and how poly(A) tails are removed by exonucleases (deadenylases), including CCR4-NOT and PAN2-PAN3. We also discuss how deadenylation rate is determined, the integration of deadenylation with other cellular processes and the function of PABPC. We conclude with an outlook for the future of research in this field.

Tuning Cell-Free Composition Controls the Time Delay, Dynamics, and Productivity of TX-TL Expression.

The composition of cell-free expression systems (TX-TL) is adjusted by adding macromolecular crowding agents and salts. However, the effects of these cosolutes on the dynamics of individual gene expression processes have not been quantified. Here, we carry out kinetic mRNA and protein level measurements on libraries of genetic constructs using the common cosolutes PEG-8000, Ficoll-400, and magnesium glutamate. By combining these measurements with biophysical modeling, we show that cosolutes have differing effects on transcription initiation, translation initiation, and translation elongation rates with trade-offs between time delays, expression tunability, and maximum expression productivity. We also confirm that biophysical models can predict translation initiation rates in TX-TL using Escherichia coli lysate. We discuss how cosolute composition can be tuned to maximize performance across different cell-free applications, including biosensing, diagnostics, and biomanufacturing.

Codon usage and protein length-dependent feedback from translation elongation regulates translation initiation and elongation speed.

Essential cellular functions require efficient production of many large proteins but synthesis of large proteins encounters many obstacles in cells. Translational control is mostly known to be regulated at the initiation step. Whether translation elongation process can feedback to regulate initiation efficiency is unclear. Codon usage bias, a universal feature of all genomes, plays an important role in determining gene expression levels. Here, we discovered that there is a conserved but codon usage-dependent genome-wide negative correlation between protein abundance and CDS length. The codon usage effects on protein expression and ribosome flux on mRNAs are influenced by CDS length; optimal codon usage preferentially promotes production of large proteins. Translation of mRNAs with long CDS and non-optimal codon usage preferentially induces phosphorylation of initiation factor eIF2α, which inhibits translation initiation efficiency. Deletion of the eIF2α kinase CPC-3 (GCN2 homolog) in Neurospora preferentially up-regulates large proteins encoded by non-optimal codons. Surprisingly, CPC-3 also inhibits translation elongation rate in a codon usage and CDS length-dependent manner, resulting in slow elongation rates for long CDS mRNAs. Together, these results revealed a codon usage and CDS length-dependent feedback mechanism from translation elongation to regulate both translation initiation and elongation kinetics.

In Vitro Reconstitution of Yeast Translation System Capable of Synthesizing Long Polypeptide and Recapitulating Programmed Ribosome Stalling.

The rates of translation elongation or termination in eukaryotes are modulated through cooperative molecular interactions involving mRNA, the ribosome, aminoacyl- and nascent polypeptidyl-tRNAs, and translation factors. To investigate the molecular mechanisms underlying these processes, we developed an in vitro translation system from yeast, reconstituted with purified translation elongation and termination factors, utilizing CrPV IGR IRES-containing mRNA, which functions in the absence of initiation factors. The system is capable of synthesizing not only short oligopeptides but also long reporter proteins such as nanoluciferase. By setting appropriate translation reaction conditions, such as the Mg2+/polyamine concentration, the arrest of translation elongation by known ribosome-stalling sequences (e.g., polyproline and CGA codon repeats) is properly recapitulated in this system. We describe protocols for the preparation of the system components, manipulation of the system, and detection of the translation products. We also mention critical parameters for setting up the translation reaction conditions. This reconstituted translation system not only facilitates biochemical analyses of translation but is also useful for various applications, such as structural and functional studies with the aim of designing drugs that act on eukaryotic ribosomes, and the development of systems for producing novel functional proteins by incorporating unnatural amino acids by eukaryotic ribosomes.

Different translation dynamics of β- and γ-actin regulates cell migration.

β- and γ-cytoplasmic actins are ubiquitously expressed in every cell type and are nearly identical at the amino acid level but play vastly different roles in vivo. Their essential roles in embryogenesis and mesenchymal cell migration critically depend on the nucleotide sequences of their genes, rather than their amino acid sequences; however, it is unclear which gene elements underlie this effect. Here we address the specific role of the coding sequence in β- and γ-cytoplasmic actins' intracellular functions, using stable polyclonal populations of immortalized mouse embryonic fibroblasts with exogenously expressed actin isoforms and their 'codon-switched' variants. When targeted to the cell periphery using β-actin 3'UTR; β-actin and γ-actin have differential effects on cell migration. These effects directly depend on the coding sequence. Single-molecule measurements of actin isoform translation, combined with fluorescence recovery after photobleaching, demonstrate a pronounced difference in β- and γ-actins' translation elongation rates in cells, leading to changes in their dynamics at focal adhesions, impairments in actin bundle formation, and reduced cell anchoring to the substrate during migration. Our results demonstrate that coding sequence-mediated differences in actin translation play a key role in cell migration.