Abstract 3660: A link between miRNAs and mRNA translation elongation: The let7-eEF2K axis in MYC-driven pediatric tumors adaptation to nutrient deprivation

Abstract Group 3 affiliation and MYC genetic amplification are associated with poor life expectancy and substantial morbidity in children suffering from medulloblastoma (MB). However, the high metabolic demand induced by MYC-driven transformation sensitizes MYC-overexpressing MB to cell death under conditions of nutrient deprivation (ND). Additionally, MYC-driven transformation is known to promote mitochondrial oxidative phosphorylation (OXPHOS). We previously reported that eukaryotic Elongation Factor Kinase 2 (eEF2K), the master regulator of mRNA translation elongation, promotes survival of MYC-overexpressing tumors under ND. Interestingly, eEF2K is overexpressed in MYC-driven MB and our preliminary proteomics data highlight large-scale alterations in OXPHOS components affecting eEF2K deficient MB cells. We therefore hypothesized that eEF2K activity is required for the selective translation of mRNAs needed for efficient OXPHOS, and for the progression of MYC-driven MB. We pefrormed Multiplexed enhanced Protein Dynamic Mass Spectrometry in eEF2K knockdown MYC-overexpressing D425 MB cells to identify mRNAs selectively translated upon eEF2K activation. Messenger RNAs encoding multiple (9 out of 10 detected) components of the mitochondrial OXPHOS pathway are selectively translated upon eEF2K activation. Inactivation of eEF2K by genetic KO leads to the disassembly of electron transport chain (ETC) complexes I-IV without affecting mRNA levels of their respective components. Consistently, eEF2K KO MB cells display decreased mitochondrial membrane potential and 20% increased proton leak thorough the mitochondrial membrane. In addition, eEF2K inactivation results in increased Group 3 MB cell death under ND and doubles survival of MB bearing mice fed with calorie restricted diets (p< 0.05). Control of mRNA translation elongation by eEF2K is critical for mitochondrial ETC complex assembly and efficient OXPHOS in MYC-overexpressing MB, likely representing an adaptive response by which MYC-driven MB cells cope with acute metabolic stress. Future therapeutic studies will aim to combine eEF2K inhibition with caloric restriction mimetic drugs as eEF2K activity appears critical under metabolic stress conditions.

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A Complementary Mechanism of Bacterial mRNA Translation Inhibition by Tetracyclines

Frontiers in Microbiology ◽

10.3389/fmicb.2021.682682 ◽

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.

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The radish Ogura fertility restorer impedes translation elongation along its cognate CMS-causing mRNA

10.1101/2021.03.17.435859 ◽

2021 ◽

Author(s):

Chuande Wang ◽

Lina Lezhneva ◽

Nadège Arnal ◽

Martine Quadrado ◽

Hakim Mireau

Keyword(s):

Mrna Translation ◽

Pollen Sterility ◽

Mitochondrial Genes ◽

Genetic Interactions ◽

Gene Control ◽

Specific Inhibition ◽

Fertility Restorer ◽

Translation Elongation ◽

Restorer Of Fertility ◽

Fertility Restorers

AbstractThe control of mRNA translation has been increasingly recognized as a key regulatory step for gene control but clear examples in eukaryotes are still scarce. Nucleo-cytoplasmic male sterilities (CMS) represent ideal genetic models to dissect genetic interactions between the mitochondria and the nucleus in plants. This trait is determined by specific mitochondrial genes and is associated with a pollen sterility phenotype that can be suppressed by nuclear genes known as restorer-of-fertility (Rf) genes. In the study, we focused on the Ogura CMS system in rapeseed and showed that the suppression to male sterility by the PPR-B fertility restorer (also called Rfo) occurs through a specific inhibition of the translation of the mitochondria-encoded CMS-causing mRNA orf138. We also demonstrate that PPR-B binds within the coding sequence of orf138 and acts as a ribosome blocker to specifically impede translation elongation along the orf138 mRNA. Rfo is the first recognized fertility restorer shown to act this way. These observations will certainly facilitate the development of synthetic fertility restorers for CMS systems in which efficient natural Rfs are lacking.

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Inferring efficiency of translation initiation and elongation from ribosome profiling

Nucleic Acids Research ◽

10.1093/nar/gkaa678 ◽

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.

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MYCN amplified neuroblastoma requires the mRNA translation regulator eEF2 kinase to adapt to nutrient deprivation

Cell Death and Differentiation ◽

10.1038/cdd.2017.79 ◽

2017 ◽

Vol 24 (9) ◽

pp. 1564-1576 ◽

Cited By ~ 13

Author(s):

Alberto Delaidelli ◽

Gian Luca Negri ◽

Asad Jan ◽

Brandon Jansonius ◽

Amal El-Naggar ◽

...

Keyword(s):

Mrna Translation ◽

Nutrient Deprivation ◽

Eef2 Kinase

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EMBR-20. ELONGATION CONTROL OF MRNA TRANSLATION DRIVES GROUP 3 MEDULLOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noab090.038 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i10-i10

Author(s):

Alberto Delaidelli ◽

Gian Luca Negri ◽

Que Xi Wang ◽

Albert Huang ◽

Simran Sidhu ◽

...

Keyword(s):

Binding Site ◽

Elongation Factor ◽

Metabolic Stress ◽

Mrna Translation ◽

Nutrient Deprivation ◽

Myc Oncogene ◽

Protein Levels ◽

Cellular Mrnas ◽

Group 3 ◽

Potential Binding Site

Abstract Medulloblastoma (MB) is the most common pediatric intracranial tumor and leading cause of childhood related cancer deaths. Group 3 affiliation and genetic amplifications of the MYC oncogene are predictors of adverse outcome in MB, underscoring a dire need for novel and more effective therapeutic approaches. The let-7 family of small non-coding RNAs (miRNAs) is known to inhibit tumor progression and regulate metabolism by targeting and degrading several cellular mRNAs, including MYC. Indeed, let-7 miRNAs are frequently repressed in several cancer types, including in MYC-driven MB. We previously reported that the mRNA translation elongation regulator eukaryotic Elongation Factor-2 Kinase (eEF2K) is a pivotal mediator of cancer cell adaptation to nutrient deprivation. In the current work, we identified a potential binding site for let-7 miRNAs on the eEF2K 3’ untranslated region (UTR). In addition, eEF2K mRNA and let-7 miRNA expressions negatively correlate in MB, suggesting a potential regulation of the former by the latter. Let-7 miRNAs transfection decreases eEF2K mRNA and protein levels (by ~40–50%). Down-regulation of luciferase activity by let-7 miRNAs is impaired upon mutation of the let-7 binding site on the eEF2K 3’UTR. Inhibition of eEF2K significantly reduces survival of MYC-amplified MB cell lines under nutrient deprivation, altering their mRNA translation rates. Knockout of eEF2K increases survival of MYC-amplified MB xenografts when mice are kept under calorie restricted diets. We conclude that let-7 miRNAs degrade the eEF2K mRNA by binding to its 3’UTR, indicating that let-7 repression in MYC-driven MB is partially responsible for increased eEF2K levels. Moreover, the let-7-eEF2K axis constitutes a critical mechanism for MYC-driven MB adaptation to acute metabolic stress, representing a promising therapeutic target. Future therapeutic studies will aim to combine eEF2K inhibition with caloric restriction mimetic drugs, as eEF2K activity appears critical under metabolic stress conditions.

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

10.1101/465914 ◽

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.

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Wobble tRNA modification and hydrophilic amino acid patterns dictate protein fate

Nature Communications ◽

10.1038/s41467-021-22254-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Francesca Rapino ◽

Zhaoli Zhou ◽

Ana Maria Roncero Sanchez ◽

Marc Joiret ◽

Christian Seca ◽

...

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Protein Expression ◽

Protein Aggregation ◽

Critical Role ◽

Mrna Translation ◽

Trna Modification ◽

Translation Elongation ◽

Hydrophilic Amino Acid ◽

Protein Output

AbstractRegulation of mRNA translation elongation impacts nascent protein synthesis and integrity and plays a critical role in disease establishment. Here, we investigate features linking regulation of codon-dependent translation elongation to protein expression and homeostasis. Using knockdown models of enzymes that catalyze the mcm5s2 wobble uridine tRNA modification (U34-enzymes), we show that gene codon content is necessary but not sufficient to predict protein fate. While translation defects upon perturbation of U34-enzymes are strictly dependent on codon content, the consequences on protein output are determined by other features. Specific hydrophilic motifs cause protein aggregation and degradation upon codon-dependent translation elongation defects. Accordingly, the combination of codon content and the presence of hydrophilic motifs define the proteome whose maintenance relies on U34-tRNA modification. Together, these results uncover the mechanism linking wobble tRNA modification to mRNA translation and aggregation to maintain proteome homeostasis.

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