scholarly journals Robustness by intrinsically disordered C-termini and translational readthrough

2018 ◽  
Vol 46 (19) ◽  
pp. 10184-10194 ◽  
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.

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

BMC Biology ◽  
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

scholarly journals An intrinsically disordered nascent protein interacts with specific regions of the ribosomal surface near the exit tunnel

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Valeria Guzman-Luna ◽  
Andrew M. Fuchs ◽  
Anna J. Allen ◽  
Alexios Staikos ◽  
Silvia Cavagnero
Keyword(s):  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Intrinsically Disordered Protein ◽  
Protein Chain ◽  
Net Charge ◽  
Weakly Interact ◽  
Intrinsically Disordered ◽  
Disordered Protein ◽  
Exit Tunnel

AbstractThe influence of the ribosome on nascent chains is poorly understood, especially in the case of proteins devoid of signal or arrest sequences. Here, we provide explicit evidence for the interaction of specific ribosomal proteins with ribosome-bound nascent chains (RNCs). We target RNCs pertaining to the intrinsically disordered protein PIR and a number of mutants bearing a variable net charge. All the constructs analyzed in this work lack N-terminal signal sequences. By a combination chemical crosslinking and Western-blotting, we find that all RNCs interact with ribosomal protein L23 and that longer nascent chains also weakly interact with L29. The interacting proteins are spatially clustered on a specific region of the large ribosomal subunit, close to the exit tunnel. Based on chain-length-dependence and mutational studies, we find that the interactions with L23 persist despite drastic variations in RNC sequence. Importantly, we also find that the interactions are highly Mg+2-concentration-dependent. This work is significant because it unravels a novel role of the ribosome, which is shown to engage with the nascent protein chain even in the absence of signal or arrest sequences.

scholarly journals Translational readthrough goes unseen by natural selection

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.

scholarly journals Identification of mRNAs that undergo stop codon readthrough in Arabidopsis thaliana

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.

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

Open Biology ◽  
2016 ◽  
Vol 6 (11) ◽  
pp. 160246 ◽  
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.

scholarly journals Evolutionary assembly patterns of prokaryotic genomes

2015 ◽  
Author(s):  
Maximilian O. Press ◽  
Christine Queitsch ◽  
Elhanan Borenstein
Keyword(s):  
Evolutionary Constraints ◽  
Historical Contingency ◽  
Phenotypic Characters ◽  
Functional Relationships ◽  
Evolutionary Innovation ◽  
Gene Level ◽  
Prokaryotic Genomes ◽  
Evolutionary Paths ◽  
Evolution Of Proteins ◽  
Sequence Substitution

AbstractEvolutionary innovation must occur in the context of some genomic background, which limits available evolutionary paths. For example, protein evolution by sequence substitution is constrained by epistasis between residues. In prokaryotes, evolutionary innovation frequently happens by macrogenomic events such as horizontal gene transfer (HGT). Previous work has suggested that HGT can be influenced by ancestral genomic content, yet the extent of such gene-level constraints has not yet been systematically characterized. Here, we evaluated the evolutionary impact of such constraints in prokaryotes, using probabilistic ancestral reconstructions from 634 extant prokaryotic genomes and a novel framework for detecting evolutionary constraints on HGT events. We identified 8,228 directional dependencies between genes, and demonstrated that many such dependencies reflect known functional relationships, including, for example, evolutionary dependencies of the photosynthetic enzyme RuBisCO. Modeling all dependencies as a network, we adapted an approach from graph theory to establish chronological precedence in the acquisition of different genomic functions. Specifically, we demonstrated that specific functions tend to be gained sequentially, suggesting that evolution in prokaryotes is governed by functional assembly patterns. Finally, we showed that these dependencies are universal rather than clade-specific and are often sufficient for predicting whether or not a given ancestral genome will acquire specific genes. Combined, our results indicate that evolutionary innovation via HGT is profoundly constrained by epistasis and historical contingency, similar to the evolution of proteins and phenotypic characters, and suggest that the emergence of specific metabolic and pathological phenotypes in prokaryotes can be predictable from current genomes.

scholarly journals High frequency of alternative first exons in erythroid genes suggests a critical role in regulating gene function

Blood ◽  
2006 ◽  
Vol 107 (6) ◽  
pp. 2557-2561 ◽  
Author(s):  
Jeff S. Tan ◽  
Narla Mohandas ◽  
John G. Conboy
Keyword(s):  
Human Genome ◽  
High Frequency ◽  
Critical Role ◽  
Test Group ◽  
Protein Isoforms ◽  
Alternative Promoters ◽  
Transcriptional Promoters ◽  
Show Evidence ◽  
Computational Analyses ◽  
Frequent Presence

AbstractThe human genome uses alternative pre-mRNA splicing as an important mechanism to encode a complex proteome from a relatively small number of genes. An unknown number of these genes also possess multiple transcriptional promoters and alternative first exons that contribute another layer of complexity to gene expression mechanisms. Using a collection of more than 100 erythroid-expressed genes as a test group, we used genome browser tools and genetic databases to assess the frequency of alternative first exons in the genome. Remarkably, 35% of these erythroid genes show evidence of alternative first exons. The majority of the candidate first exons are situated upstream of the coding exons, whereas a few are located internally within the gene. Computational analyses predict transcriptional promoters closely associated with many of the candidate first exons, supporting their authenticity. Importantly, the frequent presence of consensus translation initiation sites among the alternative first exons suggests that many proteins have alternative N-terminal structures whose expression can be coupled to promoter choice. These findings indicate that alternative promoters and first exons are more widespread in the human genome than previously appreciated and that they may play a major role in regulating expression of selected protein isoforms in a tissue-specific manner. (Blood. 2006;107: 2557-2561)

scholarly journals RPS15 mutations rewire RNA translation in chronic lymphocytic leukemia

2021 ◽  
Vol 5 (13) ◽  
pp. 2788-2792
Author(s):  
Stavroula Ntoufa ◽  
Marina Gerousi ◽  
Stamatia Laidou ◽  
Fotis Psomopoulos ◽  
Georgios Tsiolas ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Regulatory Elements ◽  
Ribosome Profiling ◽  
Lymphocytic Leukemia ◽  
Translational Efficiency ◽  
Translation Efficiency ◽  
Wild Type ◽  
Recurrent Mutations

Abstract Recent studies of chronic lymphocytic leukemia (CLL) have reported recurrent mutations in the RPS15 gene, which encodes the ribosomal protein S15 (RPS15), a component of the 40S ribosomal subunit. Despite some evidence about the role of mutant RPS15 (mostly obtained from the analysis of cell lines), the precise impact of RPS15 mutations on the translational program in primary CLL cells remains largely unexplored. Here, using RNA sequencing and ribosome profiling, a technique that involves measuring translational efficiency, we sought to obtain global insight into changes in translation induced by RPS15 mutations in CLL cells. To this end, we evaluated primary CLL cells from patients with wild-type or mutant RPS15 as well as MEC1 CLL cells transfected with mutant or wild-type RPS15. Our data indicate that RPS15 mutations rewire the translation program of primary CLL cells by reducing their translational efficiency, an effect not seen in MEC1 cells. In detail, RPS15 mutant primary CLL cells displayed altered translation efficiency of other ribosomal proteins and regulatory elements that affect key cell processes, such as the translational machinery and immune signaling, as well as genes known to be implicated in CLL, hence highlighting a relevant role for RPS15 in the natural history of CLL.

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