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 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 Stop codon readthrough of a POU transcription factor regulates steroidogenesis and developmental transitions

2020 ◽  
Author(s):  
Yunpo Zhao ◽  
Bo Gustav Lindberg ◽  
Shiva Seyedoleslami Esfahani ◽  
Xiongzhuo Tang ◽  
Stefano Piazza ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Stop Codon ◽  
Phylogenetic Analyses ◽  
Protein Isoforms ◽  
High Rate ◽  
Prothoracic Gland ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Stop Codon Readthrough

AbstractTranslational stop codon readthrough generates C-terminally extended protein isoforms. While evidence mounts of readthrough as a global phenomenon, proofs of its functional consequences are scarce. We show that readthrough of the mRNA for the Drosophila POU/Oct transcription factor Drifter occurs at a high rate and in a spatiotemporal manner in vivo, reaching above 50% in the prothoracic gland. Phylogenetic analyses suggested that readthrough of drifter is conserved among Dipterans, with C-terminal extensions harboring intrinsically disordered regions, and amino acids streches implied in transcriptional activation. The C-terminally extended Drifter isoform is required for maintaining normal levels of the growth hormone ecdysone through regulation of its biosynthetic genes, acting in synergy with the transcription factor Molting defective. A 14-bp deletion that abolished readthrough, caused prolonged larval development and delayed metamorphosis. This study provides a striking example of alternative genetic decoding that feeds into the progression from one life cycle stage to another.

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 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 Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Kotchaphorn Mangkalaphiban ◽  
Feng He ◽  
Robin Ganesan ◽  
Chan Wu ◽  
Richard Baker ◽  
...  
Keyword(s):  
Stop Codon ◽  
Regulatory Elements ◽  
Ribosome Profiling ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Coding Region ◽  
Genome Wide ◽  
A Genome ◽  
Stop Codon Readthrough

Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3′-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5′ of the stop codon, six nucleotides 3′ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3′-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3′-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 and mRNA secondary structure in the 3′-UTR had milder effects. Additionally, we found low readthrough genes to have shorter 3′-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.

scholarly journals Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae

PLoS Genetics ◽  
2021 ◽  
Vol 17 (4) ◽  
pp. e1009538
Author(s):  
Kotchaphorn Mangkalaphiban ◽  
Feng He ◽  
Robin Ganesan ◽  
Chan Wu ◽  
Richard Baker ◽  
...  
Keyword(s):  
Stop Codon ◽  
Regulatory Elements ◽  
Ribosome Profiling ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Coding Region ◽  
Genome Wide ◽  
A Genome ◽  
Stop Codon Readthrough

Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3’-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5’ of the stop codon, six nucleotides 3’ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3’-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3’-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 had milder effects. Additionally, we found low readthrough genes to have shorter 3’-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.

scholarly journals Correction: Ribosome profiling reveals pervasive and regulated stop codon readthrough in Drosophila melanogaster

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Joshua G Dunn ◽  
Catherine K Foo ◽  
Nicolette G Belletier ◽  
Elizabeth R Gavis ◽  
Jonathan S Weissman
Keyword(s):  
Drosophila Melanogaster ◽  
Stop Codon ◽  
Ribosome Profiling ◽  
Stop Codon Readthrough

scholarly journals Translational read-through promotes aggregation and shapes stop codon identity

2020 ◽  
Vol 48 (7) ◽  
pp. 3747-3760
Author(s):  
Lior Kramarski ◽  
Eyal Arbely
Keyword(s):  
Control Mechanism ◽  
Stop Codon ◽  
Release Factor ◽  
Translational Machinery ◽  
Stop Codons ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Endogenous Proteins ◽  
Quality Control Mechanism ◽  
Different Levels

Abstract Faithful translation of genetic information depends on the ability of the translational machinery to decode stop codons as termination signals. Although termination of protein synthesis is highly efficient, errors in decoding of stop codons may lead to the synthesis of C-terminally extended proteins. It was found that in eukaryotes such elongated proteins do not accumulate in cells. However, the mechanism for sequestration of C-terminally extended proteins is still unknown. Here we show that 3′-UTR-encoded polypeptides promote aggregation of the C-terminally extended proteins, and targeting to lysosomes. We demonstrate that 3′-UTR-encoded polypeptides can promote different levels of protein aggregation, similar to random sequences. We also show that aggregation of endogenous proteins can be induced by aminoglycoside antibiotics that promote stop codon read-through, by UAG suppressor tRNA, or by knokcdown of release factor 1. Furthermore, we find correlation between the fidelity of termination signals, and the predicted propensity of downstream 3′-UTR-encoded polypeptides to form intrinsically disordered regions. Our data highlight a new quality control mechanism for elimination of C-terminally elongated proteins.

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