Translational readthrough goes unseen by natural selection

Mapping Intimacies ◽

10.1101/844621 ◽

2019 ◽

Author(s):

April Snofrid Kleppe ◽

Erich Bornberg-Bauer

Keyword(s):

Protein Synthesis ◽

Natural Selection ◽

Selective Pressure ◽

Evolutionary Rate ◽

Stop Codon ◽

Gc Content ◽

Ribosome Profiling ◽

Protein Isoforms ◽

Common Error ◽

Translational Readthrough

AbstractOccasionally during protein synthesis, the ribosome bypasses the stop codon and continues translation to the next stop codon in frame. This error is called translational readthrough (TR). Earlier research suggest that TR is a relatively common error, in several taxa, yet the evolutionary relevance of this translational error is still unclear. By analysing ribosome profiling data, we have conducted species comparisons between yeasts to infer conservation of TR between orthologs. Moreover, we infer the evolutionary rate of error prone and canonically translated proteins to deduct differential selective pressure. We find that about 40% of error prone proteins in Schizosaccharomyces pombe do not have any orthologs in Saccharomyces cerevisiae, but that 60% of error prone proteins in S. pombe are undergoing canonical translation in S. cerevisiae. Error prone proteins tend to have a higher GC-content in the 3’-UTR, unlike their canonically translated ortholog. We do not find the same trends for GC-content of the CDS. We discuss the role of 3’-UTR and GC-content regarding translational readthrough. Moreover, we find that there is neither selective pressure against or for TR. We suggest that TR is a near-neutral error that goes unseen by natural selection. We speculate that TR yield neutral protein isoforms that are not being purged. We suggest that isoforms, yielded by TR, increase proteomic diversity in the cell, which is readily available upon sudden environmental shifts and which therefore may become adaptive.Author SummaryThere is an evolutionary balance act between adaptation and selection against change. Any system needs to be able to adapt facing novel environmental conditions. Simultaneously, biological systems are under selection to maintain fitness and thus undergo selection against mutations. Phenotypic mutations - translational errors during protein synthesis - have been suggested to play a role in protein evolvability by enabling quick assessment of viable phenotypes and thus enable quick adaptation. Here we test this hypothesis, by inferring evolutionary rate of proteins prone to a specific case of phenotypic mutations: translational readthrough (TR). By making use of publicly available data of yeasts, we find that TR goes unseen by natural selection and appear as a neutral event. We suggest that TR goes unseen by selection and occurs as “permissive wallflowers”, which may become relevant and yield adaptive benefits. This work highlights that stochastic processes are not necessarily under stringent selection but may prevail. In conclusion, we suggest that TR is a neutral non-adaptive process that can yield adaptive benefits.

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The functional readthrough extension of malate dehydrogenase reveals a modification of the genetic code

Open Biology ◽

10.1098/rsob.160246 ◽

2016 ◽

Vol 6 (11) ◽

pp. 160246 ◽

Cited By ~ 15

Author(s):

Julia Hofhuis ◽

Fabian Schueren ◽

Christopher Nötzel ◽

Thomas Lingner ◽

Jutta Gärtner ◽

...

Keyword(s):

Malate Dehydrogenase ◽

Stop Codon ◽

Protein Isoforms ◽

Translational Readthrough ◽

Peroxisomal Targeting ◽

Codon Context ◽

Targeting Signal ◽

Stop Codon Readthrough ◽

Insight Into ◽

Actual Ratio

Translational readthrough gives rise to C-terminally extended proteins, thereby providing the cell with new protein isoforms. These may have different properties from the parental proteins if the extensions contain functional domains. While for most genes amino acid incorporation at the stop codon is far lower than 0.1%, about 4% of malate dehydrogenase (MDH1) is physiologically extended by translational readthrough and the actual ratio of MDH1x (e x tended protein) to ‘normal' MDH1 is dependent on the cell type. In human cells, arginine and tryptophan are co-encoded by the MDH1x UGA stop codon. Readthrough is controlled by the 7-nucleotide high-readthrough stop codon context without contribution of the subsequent 50 nucleotides encoding the extension. All vertebrate MDH1x is directed to peroxisomes via a hidden peroxisomal targeting signal (PTS) in the readthrough extension, which is more highly conserved than the extension of lactate dehydrogenase B. The hidden PTS of non-mammalian MDH1x evolved to be more efficient than the PTS of mammalian MDH1x. These results provide insight into the genetic and functional co-evolution of these dually localized dehydrogenases.

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Robustness by intrinsically disordered C-termini and translational readthrough

Nucleic Acids Research ◽

10.1093/nar/gky778 ◽

2018 ◽

Vol 46 (19) ◽

pp. 10184-10194 ◽

Cited By ~ 10

Author(s):

April Snofrid Kleppe ◽

Erich Bornberg-Bauer

Keyword(s):

High Frequency ◽

Ribosomal Subunit ◽

Conformational Flexibility ◽

Ribosome Profiling ◽

Protein Isoforms ◽

Evolutionary Constraints ◽

Protein Chain ◽

Intrinsically Disordered ◽

Translational Readthrough ◽

Evolutionary Paths

Abstract During protein synthesis genetic instructions are passed from DNA via mRNA to the ribosome to assemble a protein chain. Occasionally, stop codons in the mRNA are bypassed and translation continues into the untranslated region (3′-UTR). This process, called translational readthrough (TR), yields a protein chain that becomes longer than would be predicted from the DNA sequence alone. Protein sequences vary in propensity for translational errors, which may yield evolutionary constraints by limiting evolutionary paths. Here we investigated TR in Saccharomyces cerevisiae by analysing ribosome profiling data. We clustered proteins as either prone or non-prone to TR, and conducted comparative analyses. We find that a relatively high frequency (5%) of genes undergo TR, including ribosomal subunit proteins. Our main finding is that proteins undergoing TR are highly expressed and have a higher proportion of intrinsically disordered C-termini. We suggest that highly expressed proteins may compensate for the deleterious effects of TR by having intrinsically disordered C-termini, which may provide conformational flexibility but without distorting native function. Moreover, we discuss whether minimizing deleterious effects of TR is also enabling exploration of the phenotypic landscape of protein isoforms.

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A Quick Guide to Small-Molecule Inhibitors of Eukaryotic Protein Synthesis

Biochemistry (Moscow) ◽

10.1134/s0006297920110097 ◽

2020 ◽

Vol 85 (11) ◽

pp. 1389-1421

Author(s):

S. E. Dmitriev ◽

D. O. Vladimirov ◽

K. A. Lashkevich

Keyword(s):

Protein Synthesis ◽

Small Molecule ◽

Stop Codon ◽

Ribosome Profiling ◽

Hereditary Diseases ◽

Nonsense Suppression ◽

Cell Culture Techniques ◽

Eukaryotic Protein ◽

Culture Techniques ◽

Exit Tunnel

Abstract Eukaryotic ribosome and cap-dependent translation are attractive targets in the antitumor, antiviral, anti-inflammatory, and antiparasitic therapies. Currently, a broad array of small-molecule drugs is known that specifically inhibit protein synthesis in eukaryotic cells. Many of them are well-studied ribosome-targeting antibiotics that block translocation, the peptidyl transferase center or the polypeptide exit tunnel, modulate the binding of translation machinery components to the ribosome, and induce miscoding, premature termination or stop codon readthrough. Such inhibitors are widely used as anticancer, anthelmintic and antifungal agents in medicine, as well as fungicides in agriculture. Chemicals that affect the accuracy of stop codon recognition are promising drugs for the nonsense suppression therapy of hereditary diseases and restoration of tumor suppressor function in cancer cells. Other compounds inhibit aminoacyl-tRNA synthetases, translation factors, and components of translation-associated signaling pathways, including mTOR kinase. Some of them have antidepressant, immunosuppressive and geroprotective properties. Translation inhibitors are also used in research for gene expression analysis by ribosome profiling, as well as in cell culture techniques. In this article, we review well-studied and less known inhibitors of eukaryotic protein synthesis (with the exception of mitochondrial and plastid translation) classified by their targets and briefly describe the action mechanisms of these compounds. We also present a continuously updated database (http://eupsic.belozersky.msu.ru/) that currently contains information on 370 inhibitors of eukaryotic protein synthesis.

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Stop codon readthrough alters the activity of a POU/Oct transcription factor during Drosophila development

BMC Biology ◽

10.1186/s12915-021-01106-0 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Yunpo Zhao ◽

Bo Gustav Lindberg ◽

Shiva Seyedoleslami Esfahani ◽

Xiongzhuo Tang ◽

Stefano Piazza ◽

...

Keyword(s):

Transcription Factor ◽

Larval Development ◽

Stop Codon ◽

Steroid Hormone ◽

Protein Isoforms ◽

Prothoracic Gland ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Translational Readthrough ◽

Stop Codon Readthrough

Abstract Background A number of cellular processes have evolved in metazoans that increase the proteome repertoire in relation to the genome, such as alternative splicing and translation recoding. Another such process, translational stop codon readthrough (SCR), generates C-terminally extended protein isoforms in many eukaryotes, including yeast, plants, insects, and humans. While comparative genome analyses have predicted the existence of programmed SCR in many species including humans, experimental proof of its functional consequences are scarce. Results We show that SCR of the Drosophila POU/Oct transcription factor Ventral veins lacking/Drifter (Vvl/Dfr) mRNA is prevalent in certain tissues in vivo, reaching a rate of 50% in the larval prothoracic gland. Phylogenetically, the C-terminal extension is conserved and harbors intrinsically disordered regions and amino acid stretches implied in transcriptional activation. Elimination of Vvl/Dfr translational readthrough by CRISPR/Cas9 mutagenesis changed the expression of a large number of downstream genes involved in processes such as chromatin regulation, neurogenesis, development, and immune response. As a proof-of-principle, we demonstrate that the C-terminal extension of Vvl/Dfr is necessary for correct timing of pupariation, by increasing the capacity to regulate its target genes. The extended Vvl/Dfr isoform acts in synergy with the transcription factor Molting defective (Mld) to increase the expression and biosynthesis of the steroid hormone ecdysone, thereby advancing pupariation. Consequently, late-stage larval development was prolonged and metamorphosis delayed in vvl/dfr readthrough mutants. Conclusions We demonstrate that translational recoding of a POU/Oct transcription factor takes place in a highly tissue-specific and temporally controlled manner. This dynamic and regulated recoding is necessary for normal expression of a large number of genes involved in many cellular and developmental processes. Loss of Vvl/Dfr translational readthrough negatively affects steroid hormone biosynthesis and delays larval development and progression into metamorphosis. Thus, this study demonstrates how SCR of a transcription factor can act as a developmental switch in a spatiotemporal manner, feeding into the timing of developmental transitions between different life-cycle stages. Graphical abstract

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Ribosome profiling analysis of eEF3-depleted Saccharomyces cerevisiae

10.1101/413252 ◽

2018 ◽

Author(s):

Villu Kasari ◽

Tõnu Margus ◽

Gemma C. Atkinson ◽

Marcus J.O. Johansson ◽

Vasili Hauryliuk

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Synthesis ◽

Stop Codon ◽

Elongation Factor ◽

Ribosome Profiling ◽

Open Reading Frames ◽

Additional Function ◽

Ribosome Density ◽

Eukaryotic Elongation Factor ◽

Eukaryotic Organisms

AbstractIn addition to the standard set of translation factors common in eukaryotic organisms, protein synthesis in the yeast Saccharomyces cerevisiae requires an ABCF ATPase factor eEF3, eukaryotic Elongation Factor 3. eEF3 is an E-site binder that was originally identified as an essential factor involved in the elongation stage of protein synthesis. Recent biochemical experiments suggest an additional function of eEF3 in ribosome recycling. We have characterised the global effects of eEF3 depletion on translation using ribosome profiling. Depletion of eEF3 results in decreased ribosome density at the stop codon, indicating that ribosome recycling does not become rate limiting when eEF3 levels are low. Consistent with a defect in translation elongation, eEF3 depletion causes a moderate redistribution of ribosomes towards the 5’ part of the open reading frames. We observed no E-site codon-or amino acid-specific ribosome stalling upon eEF3 depletion, supporting its role as a general elongation factor. Surprisingly, depletion of eEF3 leads to a relative decrease in P-site proline stalling, which we hypothesise is a secondary effect of generally decreased translation and/or decreased competition for the E-site with eIF5A.

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‘Stop’ in protein synthesis is modulated with exquisite subtlety by an extended RNA translation signal

Biochemical Society Transactions ◽

10.1042/bst20180190 ◽

2018 ◽

Vol 46 (6) ◽

pp. 1615-1625 ◽

Cited By ~ 9

Author(s):

Warren P. Tate ◽

Andrew G. Cridge ◽

Chris M. Brown

Keyword(s):

Protein Synthesis ◽

Conformational Changes ◽

Stop Codon ◽

Close Contact ◽

Gc Content ◽

Universal Genetic Code ◽

Rna Translation ◽

Stop Codons ◽

Release Factors ◽

Decoding Efficiency

Translational stop codons, UAA, UAG, and UGA, form an integral part of the universal genetic code. They are of significant interest today for their underlying fundamental role in terminating protein synthesis, but also for their potential utilisation for programmed alternative translation events. In diverse organisms, UAA has wide usage, but it is puzzling that the high fidelity UAG is selected against and yet UGA, vulnerable to suppression, is widely used, particularly in those archaeal and bacterial genomes with a high GC content. In canonical protein synthesis, stop codons are interpreted by protein release factors that structurally and functionally mimic decoding tRNAs and occupy the decoding site on the ribosome. The release factors make close contact with the decoding complex through multiple interactions. Correct interactions cause conformational changes resulting in new and enhanced contacts with the ribosome, particularly between specific bases in the mRNA and rRNA. The base following the stop codon (fourth or +4 base) may strongly influence decoding efficiency, facilitating alternative non-canonical events like frameshifting or selenocysteine incorporation. The fourth base is drawn into the decoding site with a compacted stop codon in the eukaryotic termination complex. Surprisingly, mRNA sequences upstream and downstream of this core tetranucleotide signal have a significant influence on the strength of the signal. Since nine bases downstream of the stop codon are within the mRNA channel, their interactions with rRNA, and r-proteins may affect efficiency. With this understanding, it is now possible to design stop signals of desired strength for specific applied purposes.

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Tracing the De Novo Origin of Protein-Coding Genes in Yeast

mBio ◽

10.1128/mbio.01024-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 9

Author(s):

Baojun Wu ◽

Alicia Knudson

Keyword(s):

Saccharomyces Cerevisiae ◽

Natural Selection ◽

De Novo ◽

Gc Content ◽

Ribosome Profiling ◽

Dna Shuffling ◽

Biological Processes ◽

Content Type ◽

New Genes ◽

Indel Mutation

ABSTRACT De novo genes are very important for evolutionary innovation. However, how these genes originate and spread remains largely unknown. To better understand this, we rigorously searched for de novo genes in Saccharomyces cerevisiae S288C and examined their spread and fixation in the population. Here, we identified 84 de novo genes in S. cerevisiae S288C since the divergence with their sister groups. Transcriptome and ribosome profiling data revealed at least 8 (10%) and 28 (33%) de novo genes being expressed and translated only under specific conditions, respectively. DNA microarray data, based on 2-fold change, showed that 87% of the de novo genes are regulated during various biological processes, such as nutrient utilization and sporulation. Our comparative and evolutionary analyses further revealed that some factors, including single nucleotide polymorphism (SNP)/indel mutation, high GC content, and DNA shuffling, contribute to the birth of de novo genes, while domestication and natural selection drive the spread and fixation of these genes. Finally, we also provide evidence suggesting the possible parallel origin of a de novo gene between S. cerevisiae and Saccharomyces paradoxus. Together, our study provides several new insights into the origin and spread of de novo genes. IMPORTANCE Emergence of de novo genes has occurred in many lineages during evolution, but the birth, spread, and function of these genes remain unresolved. Here we have searched for de novo genes from Saccharomyces cerevisiae S288C using rigorous methods, which reduced the effects of bad annotation and genomic gaps on the identification of de novo genes. Through this analysis, we have found 84 new genes originating de novo from previously noncoding regions, 87% of which are very likely involved in various biological processes. We noticed that 10% and 33% of de novo genes were only expressed and translated under specific conditions, therefore, verification of de novo genes through transcriptome and ribosome profiling, especially from limited expression data, may underestimate the number of bona fide new genes. We further show that SNP/indel mutation, high GC content, and DNA shuffling could be involved in the birth of de novo genes, while domestication and natural selection drive the spread and fixation of these genes. Finally, we provide evidence suggesting the possible parallel origin of a new gene.

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Identification of mRNAs that undergo stop codon readthrough in Arabidopsis thaliana

10.1101/2021.11.09.467898 ◽

2021 ◽

Author(s):

Sarthak Sahoo ◽

Divyoj Singh ◽

Anumeha Singh ◽

Sandeep M. Eswarappa

Keyword(s):

Arabidopsis Thaliana ◽

Stop Codon ◽

Protein Localization ◽

Abiotic Stress Tolerance ◽

Ribosome Profiling ◽

Protein Isoforms ◽

Functional Enrichment ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Stop Codon Readthrough

A stop codon ensures termination of translation at a specific position on an mRNA. Sometimes, termination fails as translation machinery recognizes a stop codon as a sense codon. This leads to stop codon readthrough (SCR) resulting in the continuation of translation beyond the stop codon, generating protein isoforms with C-terminal extension. SCR has been observed in viruses, fungi, and multicellular organisms including mammals. However, SCR is largely unexplored in plants. In this study, we have analyzed ribosome profiling datasets to identify mRNAs that undergo SCR in Arabidopsis thaliana. Analyses of the ribosome density, ribosome coverage and three-nucleotide periodicity of the ribosome profiling reads, in the mRNA region downstream of the stop codon, provided strong evidence for SCR in mRNAs of 144 genes. This process generates putative peroxisomal targeting signal, nuclear localization signal, prenylation signal, transmembrane helix and intrinsically disordered regions in the C-terminal extension of several of these proteins. Gene ontology (GO) functional enrichment analysis revealed that these 144 genes belong to three major functional groups - translation, photosynthesis and abiotic stress tolerance. Finally, using a luminescence-based assay, we experimentally demonstrate SCR in representative mRNAs belonging to these functional classes. Based on these observations, we propose that SCR plays an important role in plant physiology by regulating the protein localization and function.

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