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

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.

Download Full-text

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

Download Full-text

Peroxisomal lactate dehydrogenase is generated by translational readthrough in mammals

eLife ◽

10.7554/elife.03640 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 89

Author(s):

Fabian Schueren ◽

Thomas Lingner ◽

Rosemol George ◽

Julia Hofhuis ◽

Corinna Dickel ◽

...

Keyword(s):

Lactate Dehydrogenase ◽

In Silico ◽

Stop Codon ◽

The Other ◽

Translational Readthrough ◽

Peroxisomal Targeting ◽

Signal Type ◽

Targeting Signal ◽

Peroxisomal Targeting Signal

Translational readthrough gives rise to low abundance proteins with C-terminal extensions beyond the stop codon. To identify functional translational readthrough, we estimated the readthrough propensity (RTP) of all stop codon contexts of the human genome by a new regression model in silico, identified a nucleotide consensus motif for high RTP by using this model, and analyzed all readthrough extensions in silico with a new predictor for peroxisomal targeting signal type 1 (PTS1). Lactate dehydrogenase B (LDHB) showed the highest combined RTP and PTS1 probability. Experimentally we show that at least 1.6% of the total cellular LDHB is targeted to the peroxisome by a conserved hidden PTS1. The readthrough-extended lactate dehydrogenase subunit LDHBx can also co-import LDHA, the other LDH subunit, into peroxisomes. Peroxisomal LDH is conserved in mammals and likely contributes to redox equivalent regeneration in peroxisomes.

Download Full-text

Translational readthrough goes unseen by natural selection

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.

Download Full-text

Evolutionary dynamics of abundant stop codon readthrough inAnophelesandDrosophila

10.1101/051557 ◽

2016 ◽

Author(s):

Irwin Jungreis ◽

Clara S Chan ◽

Robert M Waterhouse ◽

Gabriel Fields ◽

Michael F Lin ◽

...

Keyword(s):

Evolutionary Dynamics ◽

Stop Codon ◽

Rna Structures ◽

Stop Codons ◽

New Genes ◽

Single Clade ◽

Peroxisomal Targeting ◽

Recent Developments ◽

Yeast Genes ◽

Stop Codon Readthrough

AbstractTranslational stop codon readthrough was virtually unknown in eukaryotic genomes until recent developments in comparative genomics and new experimental techniques revealed evidence of readthrough in hundreds of fly genes and several human, worm, and yeast genes. Here, we use the genomes of 21 species ofAnophelesmosquitoes and improved comparative techniques to identify evolutionary signatures of conserved, functional readthrough of 353 stop codons in the malaria vector,Anopheles gambiae, and 51 additionalDrosophila melanogasterstop codons, with several cases of double and triple readthrough including readthrough of two adjacent stop codons, supporting our earlier prediction of abundant readthrough in pancrustacea genomes. Comparisons betweenAnophelesandDrosophilaallow us to transcend the static picture provided by single-clade analysis to explore the evolutionary dynamics of abundant readthrough. We find that most differences between the readthrough repertoires of the two species are due to readthrough gain or loss in existing genes, rather than to birth of new genes or to gene death; that RNA structures are sometimes gained or lost while readthrough persists; and that readthrough is more likely to be lost at TAA and TAG stop codons. We also determine which characteristic properties of readthrough predate readthrough and which are clade-specific. We estimate that there are more than 600 functional readthrough stop codons inA. gambiaeand 900 inD. melanogaster. We find evidence that readthrough is used to regulate peroxisomal targeting in two genes. Finally, we use the sequenced centipede genome to refine the phylogenetic extent of abundant readthrough.

Download Full-text

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

10.1101/2020.05.30.125401 ◽

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.

Download Full-text

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.

Download Full-text

Stop Codon Context-Specific Induction of Translational Readthrough

Biomolecules ◽

10.3390/biom11071006 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1006

Author(s):

Mirco Schilff ◽

Yelena Sargsyan ◽

Julia Hofhuis ◽

Sven Thoms

Keyword(s):

Premature Termination ◽

Stop Codon ◽

Premature Termination Codon ◽

Termination Codon ◽

Reporter System ◽

Pathogenic Variants ◽

Drug Concentrations ◽

Translational Readthrough ◽

Monogenic Diseases ◽

Codon Context

Premature termination codon (PTC) mutations account for approximately 10% of pathogenic variants in monogenic diseases. Stimulation of translational readthrough, also known as stop codon suppression, using translational readthrough-inducing drugs (TRIDs) may serve as a possible therapeutic strategy for the treatment of genetic PTC diseases. One important parameter governing readthrough is the stop codon context (SCC) – the stop codon itself and the nucleotides in the vicinity of the stop codon on the mRNA. However, the quantitative influence of the SCC on treatment outcome and on appropriate drug concentrations are largely unknown. Here, we analyze the readthrough-stimulatory effect of various readthrough-inducing drugs on the SCCs of five common premature termination codon mutations of PEX5 in a sensitive dual reporter system. Mutations in PEX5, encoding the peroxisomal targeting signal 1 receptor, can cause peroxisomal biogenesis disorders of the Zellweger spectrum. We show that the stop context has a strong influence on the levels of readthrough stimulation and impacts the choice of the most effective drug and its concentration. These results highlight potential advantages and the personalized medicine nature of an SCC-based strategy in the therapy of rare diseases.

Download Full-text

Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100157760 ◽

1990 ◽

Vol 48 (3) ◽

pp. 44-45

Author(s):

G-A. Keller ◽

S. J. Gould ◽

S. Subramani ◽

S. Krisans

Keyword(s):

Mammalian Cells ◽

Molecular Mechanisms ◽

Protein Targeting ◽

Firefly Luciferase ◽

Peroxisomal Enzyme ◽

Transfected Cells ◽

Peroxisomal Targeting ◽

Targeting Signal ◽

Series Of Experiments ◽

Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

Download Full-text