scholarly journals Organ-specific translation elongation rates measured by in vivo ribosome profiling

2018 ◽  
Author(s):  
Maxim V. Gerashchenko ◽  
Zalan Peterfi ◽  
Vadim N. Gladyshev
Keyword(s):  
Protein Synthesis ◽  
Translation Initiation ◽  
Ribosome Profiling ◽  
Dietary Interventions ◽  
Translation Elongation ◽  
Time Resolved ◽  
Gene Level ◽  
Organ Specific ◽  
Cell Type Specific

Protein synthesis and degradation are intricate biological processes involving more than a hundred proteins operating in a highly orches-trated fashion. Despite the progress, few options are available to access translation in live animals as the increase in animal’s complexity limits the repertoire of experimental tools that could be applied to observe and manipulate processes within animal’s body, organs, and individual cells. It this study, we developed a labeling-free method for measuring organ- and cell-type specific translation elongation rates. It is based on a 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 among mouse organs and determined them to be 6.8, 5.2, and 4.4 amino acids per sec for liver, kidney, and skeletal muscle, respectively.SignificanceProtein synthesis is a vital biological process. Modern methods of genome editing enable generation of sophisticated animal models to study the regulation of protein synthesis in health end disease. However, the methods that could track various steps of translation at a gene level resolution in vivo are lacking, particularly in complex vertebrates, such as mice and rats. Here, we measured the translation elongation rate in several organs by delivering inhibitors specific to certain phases of translation directly through the mouse bloodstream. This study lays out a path for interrogating translation in animals in response to various genetic and dietary interventions.

scholarly journals Translation elongation rate varies among organs and decreases with age

2020 ◽  
Author(s):  
Maxim V Gerashchenko ◽  
Zalan Peterfi ◽  
Sun Hee Yim ◽  
Vadim N Gladyshev
Keyword(s):  
Translation Initiation ◽  
Elongation Rate ◽  
Ribosome Profiling ◽  
Translation Elongation ◽  
Time Resolved ◽  
Specific Manner ◽  
Organ Specific ◽  
Cell Type Specific ◽  
Polypeptide Chains

Abstract 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.

scholarly journals Inferring efficiency of translation initiation and elongation from ribosome profiling

2020 ◽  
Vol 48 (17) ◽  
pp. 9478-9490
Author(s):  
Juraj Szavits-Nossan ◽  
Luca Ciandrini
Keyword(s):  
Protein Synthesis ◽  
Mathematical Modelling ◽  
Translation Initiation ◽  
Mrna Translation ◽  
Elongation Rate ◽  
Ribosome Profiling ◽  
Translation Elongation ◽  
Recent Advances ◽  
Two Stages

Abstract One of the main goals of ribosome profiling is to quantify the rate of protein synthesis at the level of translation. Here, we develop a method for inferring translation elongation kinetics from ribosome profiling data using recent advances in mathematical modelling of mRNA translation. Our method distinguishes between the elongation rate intrinsic to the ribosome’s stepping cycle and the actual elongation rate that takes into account ribosome interference. This distinction allows us to quantify the extent of ribosomal collisions along the transcript and identify individual codons where ribosomal collisions are likely. When examining ribosome profiling in yeast, we observe that translation initiation and elongation are close to their optima and traffic is minimized at the beginning of the transcript to favour ribosome recruitment. However, we find many individual sites of congestion along the mRNAs where the probability of ribosome interference can reach $50\%$. Our work provides new measures of translation initiation and elongation efficiencies, emphasizing the importance of rating these two stages of translation separately.

scholarly journals Inferring efficiency of translation initiation and elongation from ribosome profiling

2019 ◽  
Author(s):  
Juraj Szavits-Nossan ◽  
Luca Ciandrini
Keyword(s):  
Protein Synthesis ◽  
Mathematical Modelling ◽  
Translation Initiation ◽  
Mrna Translation ◽  
Elongation Rate ◽  
Ribosome Profiling ◽  
Translation Elongation ◽  
Recent Advances ◽  
Two Stages

ABSTRACTOne of the main goals of ribosome profiling is to quantify the rate of protein synthesis at the level of translation. Here, we develop a method for inferring translation elongation kinetics from ribosome profiling data using recent advances in the mathematical modelling of mRNA translation. Our method distinguishes between the elongation rate intrinsic to the ribosome’s stepping cycle and the actual elongation rate that takes into account ribosome interference. This distinction allows us to quantify the extent of ribosomal collisions along the transcript and identify individual codons where ribosomal collisions are likely. When examining ribosome profiling in yeast, we observe that translation initiation and elongation are close to their optima, and traffic is minimised at the beginning of the transcript to favour ribosome recruitment. However, we find many individual sites of congestion along the mRNAs where the probability of ribosome interference can reach 50%. Our work provides new measures of translation initiation and elongation efficiencies, emphasising the importance of rating these two stages of translation separately.

scholarly journals A Complementary Mechanism of Bacterial mRNA Translation Inhibition by Tetracyclines

2021 ◽  
Vol 12 ◽  
Author(s):  
Victor Barrenechea ◽  
Maryhory Vargas-Reyes ◽  
Miguel Quiliano ◽  
Pohl Milón
Keyword(s):  
Protein Synthesis ◽  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Mrna Translation ◽  
Resistance Mechanisms ◽  
Dissociation Rate ◽  
Initiation Factor ◽  
Translation Elongation ◽  
Initiation Phase ◽  
Complementary Mechanism

Tetracycline has positively impacted human health as well as the farming and animal industries. Its extensive usage and versatility led to the spread of resistance mechanisms followed by the development of new variants of the antibiotic. Tetracyclines inhibit bacterial growth by impeding the binding of elongator tRNAs to the ribosome. However, a small number of reports indicated that Tetracyclines could also inhibit translation initiation, yet the molecular mechanism remained unknown. Here, we use biochemical and computational methods to study how Oxytetracycline (Otc), Demeclocycline (Dem), and Tigecycline (Tig) affect the translation initiation phase of protein synthesis. Our results show that all three Tetracyclines induce Initiation Factor IF3 to adopt a compact conformation on the 30S ribosomal subunit, similar to that induced by Initiation Factor IF1. This compaction was faster for Tig than Dem or Otc. Furthermore, all three tested tetracyclines affected IF1-bound 30S complexes. The dissociation rate constant of IF1 in early 30S complexes was 14-fold slower for Tig than Dem or Otc. Late 30S initiation complexes (30S pre-IC or IC) exhibited greater IF1 stabilization by Tig than for Dem and Otc. Tig and Otc delayed 50S joining to 30S initiation complexes (30S ICs). Remarkably, the presence of Tig considerably slowed the progression to translation elongation and retained IF1 in the resulting 70S initiation complex (70S IC). Molecular modeling of Tetracyclines bound to the 30S pre-IC and 30S IC indicated that the antibiotics binding site topography fluctuates along the initiation pathway. Mainly, 30S complexes show potential contacts between Dem or Tig with IF1, providing a structural rationale for the enhanced affinity of the antibiotics in the presence of the factor. Altogether, our data indicate that Tetracyclines inhibit translation initiation by allosterically perturbing the IF3 layout on the 30S, retaining IF1 during 70S IC formation, and slowing the transition toward translation elongation. Thus, this study describes a new complementary mechanism by which Tetracyclines may inhibit bacterial protein synthesis.

scholarly journals Translation elongation factor 2 depletion by siRNA in mouse liver leads to mTOR-independent translational upregulation of ribosomal protein genes

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Maxim V. Gerashchenko ◽  
Mikhail V. Nesterchuk ◽  
Elena M. Smekalova ◽  
Joao A. Paulo ◽  
Piotr S. Kowalski ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Ribosomal Proteins ◽  
Mouse Liver ◽  
Negative Impact ◽  
Elongation Factor ◽  
Ribosome Profiling ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Elongation Factor 2

Abstract Due to breakthroughs in RNAi and genome editing methods in the past decade, it is now easier than ever to study fine details of protein synthesis in animal models. However, most of our understanding of translation comes from unicellular organisms and cultured mammalian cells. In this study, we demonstrate the feasibility of perturbing protein synthesis in a mouse liver by targeting translation elongation factor 2 (eEF2) with RNAi. We were able to achieve over 90% knockdown efficacy and maintain it for 2 weeks effectively slowing down the rate of translation elongation. As the total protein yield declined, both proteomics and ribosome profiling assays showed robust translational upregulation of ribosomal proteins relative to other proteins. Although all these genes bear the TOP regulatory motif, the branch of the mTOR pathway responsible for translation regulation was not activated. Paradoxically, coordinated translational upregulation of ribosomal proteins only occurred in the liver but not in murine cell culture. Thus, the upregulation of ribosomal transcripts likely occurred via passive mTOR-independent mechanisms. Impaired elongation sequesters ribosomes on mRNA and creates a shortage of free ribosomes. This leads to preferential translation of transcripts with high initiation rates such as ribosomal proteins. Furthermore, severe eEF2 shortage reduces the negative impact of positively charged amino acids frequent in ribosomal proteins on ribosome progression.

scholarly journals The alarmones (p)ppGpp directly regulate translation initiation during entry into quiescence

2020 ◽  
Vol 117 (27) ◽  
pp. 15565-15572 ◽  
Author(s):  
Simon Diez ◽  
Jaewook Ryu ◽  
Kelvin Caban ◽  
Ruben L. Gonzalez ◽  
Jonathan Dworkin
Keyword(s):  
Protein Synthesis ◽  
Translation Initiation ◽  
Direct Interaction ◽  
Extended Period ◽  
Initiation Factor ◽  
Synthetic Activity ◽  
Inhibit Protein Synthesis ◽  
Low Protein ◽  
Initiation Factor 2

Many bacteria exist in a state of metabolic quiescence where energy consumption must be minimized so as to maximize available resources over a potentially extended period of time. As protein synthesis is the most energy intensive metabolic process in a bacterial cell, it would be an appropriate target for down-regulation during the transition from growth to quiescence. We observe that whenBacillus subtilisexits rapid growth, a subpopulation of cells emerges with very low protein synthetic activity. This phenotypic heterogeneity requires the production of the nucleotides (p)ppGpp, which we show are sufficient to inhibit protein synthesis in vivo. We then show that one of these molecules, ppGpp, inhibits protein synthesis by preventing the allosteric activation of the essential GTPase Initiation Factor 2 (IF2) during translation initiation. Finally, we demonstrate that the observed attenuation of protein synthesis during the entry into quiescence is a consequence of the direct interaction of (p)ppGpp and IF2.

scholarly journals Fidelity of translation initiation is required for coordinated respiratory complex assembly

2019 ◽  
Vol 5 (12) ◽  
pp. eaay2118 ◽  
Author(s):  
Danielle L. Rudler ◽  
Laetitia A. Hughes ◽  
Kara L. Perks ◽  
Tara R. Richman ◽  
Irina Kuznetsova ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Mitochondrial Protein ◽  
Molecular Machines ◽  
Ribosome Profiling ◽  
Untranslated Regions ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Translation ◽  
Initiation Factors ◽  
Mitochondrial Ribosomes

Mammalian mitochondrial ribosomes are unique molecular machines that translate 11 leaderless mRNAs; however, it is not clear how mitoribosomes initiate translation, since mitochondrial mRNAs lack untranslated regions. Mitochondrial translation initiation shares similarities with prokaryotes, such as the formation of a ternary complex of fMet-tRNAMet, mRNA and the 28S subunit, but differs in the requirements for initiation factors. Mitochondria have two initiation factors: MTIF2, which closes the decoding center and stabilizes the binding of the fMet-tRNAMet to the leaderless mRNAs, and MTIF3, whose role is not clear. We show that MTIF3 is essential for survival and that heart- and skeletal muscle–specific loss of MTIF3 causes cardiomyopathy. We identify increased but uncoordinated mitochondrial protein synthesis in mice lacking MTIF3, resulting in loss of specific respiratory complexes. Ribosome profiling shows that MTIF3 is required for recognition and regulation of translation initiation of mitochondrial mRNAs and for coordinated assembly of OXPHOS complexes in vivo.

Dominant-Negative Inhibition of MCT-1 Oncogene Attenuates a Malignant Phenotype through Translational Repression.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4168-4168
Author(s):  
Patrick Hagner ◽  
Krystyna Mazan-Mamczarz ◽  
Sharon Corl ◽  
Ron B. Gartenhaus
Keyword(s):  
Protein Synthesis ◽  
Cell Lines ◽  
Translation Initiation ◽  
Deletion Mutant ◽  
Cell Lymphoma ◽  
Dominant Negative ◽  
Translational Repression ◽  
Malignant Phenotype ◽  
Empty Vector

Abstract Background: Gene expression is controlled at multiple levels. Translation initiation is a critical checkpoint for regulating levels of protein synthesis. The MCT-1 (Multiple copies in T-cell lymphoma 1) oncogene product has been shown to bind to the cap complex through its PUA domain and recruit DENR, a SUI1 motif containing protein that can increase translation initiation of target mRNAs by scanning and recognition of the initiation codon. An important function of MCT-1 is its modulation of a subset of cancer related mRNAs in human tumors. Levels of MCT-1 protein are increased in a number of non-Hodgkin’s lymphoma cell lines and diffuse large B-cell lymphoma. The abnormal regulation of protein synthesis in lymphoma cells has the potential to be exploited for cancer therapy. We have shown that an MCT-1 deletion mutant, containing only the PUA domain interacts with the cap-complex but does not promote translation. We hypothesized that a PUA-domain mutant acting as dominant-negative would interfere with MCT-1 function through translational repression and modify the malignant phenotype. Method: Using a retroviral vector we established stable Jurkat cell lines expressing either PUA (Jurkat-PUA) or empty vector (Jurkat-V) in order to investigate the feasibility of targeting MCT-1 using a dominant-negative approach and its impact on the transformed phenotype. Multiple clones were plated in soft agar to determine anchorage-independent growth capacity. We also examined growth under reduced serum conditions to evaluate growth kinetics under stress conditions. Jurkat cell lines expressing either PUA or empty vector were exposed to either doxorubicin or gamma-irradiation and cell viability was assessed using both trypan blue exclusion and TUNEL assay. Effects on translation were assayed employing a combination of Western blotting and in-vivo translation assays. Results: There was a greater than three-fold difference in colony formation comparing Jurkat-V with Jurkat-PUA cells. Under serum deprivation conditions Jurkat-PUA grew much slower than Jurkat-V, and cell cycle analysis demonstrated Jurkat-PUA clones progressing through the cell cycle significantly slower than Jurkat-V clones. Sensitivity to both doxorubicin and gamma-radiation was increased at least 2-fold in cells expressing the PUA deletion mutant. Quantitative real-time PCR performed in Jurkat-V and Jurkat-PUA cells demonstrated equivalent levels of selected target mRNAs including Dp-1 and Cyclin D1 however, there were lower protein levels in the Jurkat-PUA clones. Finally, Jurkat-PUA cells displayed reduced in-vivo translation. Conclusion: We have shown that Jurkat cells retrovirally transduced with a dominant-negative MCT-1 mutant can interfere with protein synthesis and modify the malignant phenotype of a highly aggressive lymphoma. As proof of principle, we have established the utility of targeting MCT-1 and the translation initiation complex in lymphoma cells as a potentially useful therapeutic approach.

scholarly journals Protein synthesis rates and ribosome occupancies reveal determinants of translation elongation rates

2018 ◽  
Author(s):  
Andrea Riba ◽  
Noemi Di Nanni ◽  
Nitish Mittal ◽  
Erik Arhné ◽  
Alexander Schmidt ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Translation Initiation ◽  
Mrna Translation ◽  
Elongation Rate ◽  
Translation Elongation ◽  
Lower Protein ◽  
Protein Output

AbstractAlthough protein synthesis dynamics has been studied both with theoretical models and by profiling ribosome footprints, the determinants of ribosome flux along open reading frames (ORFs) are not fully understood. Combining measurements of protein synthesis rate with ribosome footprinting data, we here inferred translation initiation and elongation rates for over a thousand ORFs in exponentially-growing wildtype yeast cells. We found that the amino acid composition of synthesized proteins is as important a determinant of translation elongation rate as parameters related to codon and tRNA adaptation. We did not find evidence of ribosome collisions curbing the protein output of yeast transcripts, either in high translation conditions associated with exponential growth, or in strains in which deletion of individual ribosomal protein genes leads to globally increased or decreased translation. Slow translation elongation is characteristic of RP-encoding transcripts, which have markedly lower protein output than other transcripts with equally high ribosome densities.Significance StatementAlthough sequencing of ribosome footprints has uncovered new aspects of mRNA translation, the determinants of ribosome flux remain incompletely understood. Combining ribosome footprint data with measurements of protein synthesis rates, we here inferred translation initiation and elongation rates for over a thousand ORFs in yeast strains with varying translation capacity. We found that the translation elongation rate varies up to ~20-fold among yeast transcripts, and is significantly correlated with the rate of translation initiation. Furthermore, the amino acid composition of synthesized proteins impacts the rate of translation elongation to the same extent as measures of codon and tRNA adaptation. Transcripts encoding ribosomal proteins are translated especially slow, having markedly lower protein output than other transcripts with equally high ribosome densities.

scholarly journals Quantitative Modeling of Protein Synthesis Using Ribosome Profiling Data

2021 ◽  
Vol 8 ◽  
Author(s):  
Vandana Yadav ◽  
Inayat Ullah Irshad ◽  
Hemant Kumar ◽  
Ajeet K. Sharma
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Translation Initiation ◽  
Regulation Of Gene Expression ◽  
Ribosome Profiling ◽  
Quantitative Modeling ◽  
Quantitative Prediction ◽  
Translation Rate ◽  
The Past

Quantitative prediction on protein synthesis requires accurate translation initiation and codon translation rates. Ribosome profiling data, which provide steady-state distribution of relative ribosome occupancies along a transcript, can be used to extract these rate parameters. Various methods have been developed in the past few years to measure translation-initiation and codon translation rates from ribosome profiling data. In the review, we provide a detailed analysis of the key methods employed to extract the translation rate parameters from ribosome profiling data. We further discuss how these approaches were used to decipher the role of various structural and sequence-based features of mRNA molecules in the regulation of gene expression. The utilization of these accurate rate parameters in computational modeling of protein synthesis may provide new insights into the kinetic control of the process of gene expression.

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