Decision letter: Stop codon context influences genome-wide stimulation of termination codon readthrough by aminoglycosides

AbstractStop codon readthrough (SCR) occurs when the ribosome miscodes at a stop codon. Such readthrough events can be therapeutically desirable when a premature termination codon (PTC) is found in a critical gene. To study SCR in vivo in a genome-wide manner, we treated mammalian cells with aminoglycosides and performed ribosome profiling. We find that in addition to stimulating readthrough of PTCs, aminoglycosides stimulate readthrough of normal termination codons (NTCs) genome-wide. Stop codon identity, the nucleotide following the stop codon, and the surrounding mRNA sequence context all influence the likelihood of SCR. In comparison to NTCs, downstream stop codons in 3′UTRs are recognized less efficiently by ribosomes, suggesting that targeting of critical stop codons for readthrough may be achievable without general disruption of translation termination. Finally, we find that G418 treatment globally alters gene expression with substantial effects on translation of histone genes, selenoprotein genes, and S-adenosylmethionine decarboxylase (AMD1).

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Stop codon context influences genome-wide stimulation of termination codon readthrough by aminoglycosides

eLife ◽

10.7554/elife.52611 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 19

Author(s):

Jamie R Wangen ◽

Rachel Green

Keyword(s):

Mammalian Cells ◽

Stop Codon ◽

Translation Termination ◽

Premature Termination Codon ◽

Ribosome Profiling ◽

Termination Codon ◽

Stop Codons ◽

Genome Wide ◽

A Genome

Stop codon readthrough (SCR) occurs when the ribosome miscodes at a stop codon. Such readthrough events can be therapeutically desirable when a premature termination codon (PTC) is found in a critical gene. To study SCR in vivo in a genome-wide manner, we treated mammalian cells with aminoglycosides and performed ribosome profiling. We find that in addition to stimulating readthrough of PTCs, aminoglycosides stimulate readthrough of normal termination codons (NTCs) genome-wide. Stop codon identity, the nucleotide following the stop codon, and the surrounding mRNA sequence context all influence the likelihood of SCR. In comparison to NTCs, downstream stop codons in 3′UTRs are recognized less efficiently by ribosomes, suggesting that targeting of critical stop codons for readthrough may be achievable without general disruption of translation termination. Finally, we find that G418-induced miscoding alters gene expression with substantial effects on translation of histone genes, selenoprotein genes, and S-adenosylmethionine decarboxylase (AMD1).

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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.

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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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MORFEE: a new tool for detecting and annotating single nucleotide variants creating premature ATG codons from VCF files

10.1101/2020.03.29.012054 ◽

2020 ◽

Cited By ~ 1

Author(s):

Dylan Aïssi ◽

Omar Soukarieh ◽

Carole Proust ◽

Beatrice Jaspard-Vinassa ◽

Pierre Fautrad ◽

...

Keyword(s):

Stop Codon ◽

Association Studies ◽

Premature Stop Codon ◽

Open Reading Frames ◽

Strong Impact ◽

Genome Wide Association Studies ◽

Single Nucleotide Variants ◽

Functional Variants ◽

Genome Wide ◽

Upstream Open Reading Frames

AbstractSummaryVariants in 5’UTR regions that create upstream translation initiation AUG codons are a class of neglected non coding variations. When they associate with a premature stop codon and create upstream open reading frames (uORFs) whose translation competes with that of natural proteins, they can have strong impact on human diseases. We here describe MORFEE, a new bioinformatics tool that detects, annotates and predicts, from a standard VCF file, the creation of uORF by any 5’UTR variants on uORF creation. MORFEE was applied to two genomic resources and identified candidate functional variants that could explain statistical association signals observed in the context of Genome Wide Association Studies or could be responsible for rare forms of diseases. In conclusion MORFEE is an easy-to-use tool complementary to existing ones that can help resolving genetic investigations that remained so far unfruitful.Availability and implementationMORFEE is written in R with code and package available at https://github.com/daissi/[email protected]; [email protected]

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Genome-wide effects of the antimicrobial peptide apidaecin on translation termination in bacteria

eLife ◽

10.7554/elife.62655 ◽

2020 ◽

Vol 9 ◽

Author(s):

Kyle Mangano ◽

Tanja Florin ◽

Xinhao Shao ◽

Dorota Klepacki ◽

Irina Chelysheva ◽

...

Keyword(s):

Antimicrobial Peptide ◽

Stop Codon ◽

Translation Termination ◽

Bacterial Cells ◽

Release Factor ◽

Translation Arrest ◽

Genome Wide Analysis ◽

Genome Wide ◽

Exit Tunnel ◽

Unique Mechanism

Biochemical studies suggested that the antimicrobial peptide apidaecin (Api) inhibits protein synthesis by binding in the nascent peptide exit tunnel and trapping the release factor associated with a terminating ribosome. The mode of Api action in bacterial cells had remained unknown. Here genome-wide analysis reveals that in bacteria, Api arrests translating ribosomes at stop codons and causes pronounced queuing of the trailing ribosomes. By sequestering the available release factors, Api promotes pervasive stop codon bypass, leading to the expression of proteins with C-terminal extensions. Api-mediated translation arrest leads to the futile activation of the ribosome rescue systems. Understanding the unique mechanism of Api action in living cells may facilitate the development of new medicines and research tools for genome exploration.

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Genome-wide prediction of stop codon readthrough during translation in the yeast Saccharomyces cerevisiae

Nucleic Acids Research ◽

10.1093/nar/gkh1004 ◽

2004 ◽

Vol 32 (22) ◽

pp. 6605-6616 ◽

Cited By ~ 50

Author(s):

I. Williams

Keyword(s):

Saccharomyces Cerevisiae ◽

Stop Codon ◽

Yeast Saccharomyces Cerevisiae ◽

Genome Wide ◽

Stop Codon Readthrough

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Effects of an Opal Termination Codon Preceding the nsP4 Gene Sequence in the O'Nyong-Nyong Virus Genome on Anopheles gambiae Infectivity

Journal of Virology ◽

10.1128/jvi.80.10.4992-4997.2006 ◽

2006 ◽

Vol 80 (10) ◽

pp. 4992-4997 ◽

Cited By ~ 33

Author(s):

Kevin M. Myles ◽

Cindy L. H. Kelly ◽

Jeremy P. Ledermann ◽

Ann M. Powers

Keyword(s):

Anopheles Gambiae ◽

De Novo ◽

Stop Codon ◽

Virus Genome ◽

Termination Codon ◽

De Novo Mutation ◽

Nonstructural Proteins ◽

Reading Frame ◽

Fitness Advantage ◽

Translational Readthrough

ABSTRACT The genomic RNA of an alphavirus encodes four different nonstructural proteins, nsP1, nsP2, nsP3, and nsP4. The polyprotein P123 is produced when translation terminates at an opal termination codon between nsP3 and nsP4. The polyprotein P1234 is produced when translational readthrough occurs or when the opal termination codon has been replaced by a sense codon in the alphavirus genome. Evolutionary pressures appear to have maintained genomic sequences encoding both a stop codon (opal) and an open reading frame (arginine) as a general feature of the O'nyong-nyong virus (ONNV) genome, indicating that both are required at some point. Alternate replication of ONNVs in both vertebrate and invertebrate hosts may determine predominance of a particular codon at this locus in the viral quasispecies. However, no systematic study has previously tested this hypothesis in whole animals. We report here the results of the first study to investigate in a natural mosquito host the functional significance of the opal stop codon in an alphavirus genome. We used a full-length cDNA clone of ONNV to construct a series of mutants in which the arginine between nsP3 and nsP4 was replaced with an opal, ochre, or amber stop codon. The presence of an opal stop codon upstream of nsP4 nearly doubled (75.5%) the infectivity of ONNV over that of virus possessing a codon for the amino acid arginine at the corresponding position (39.8%). Although the frequency with which the opal virus disseminated from the mosquito midgut did not differ significantly from that of the arginine virus on days 8 and 10, dissemination did began earlier in mosquitoes infected with the opal virus. Although a clear fitness advantage is provided to ONNV by the presence of an opal codon between nsP3 and nsP4 in Anopheles gambiae, sequence analysis of ONNV RNA extracted from mosquito bodies and heads indicated codon usage at this position corresponded with that of the virus administered in the blood meal. These results suggest that while selection of ONNV variants is occurring, de novo mutation at the position between nsP3 and nsP4 does not readily occur in the mosquito. Taken together, these results suggest that the primary fitness advantage provided to ONNV by the presence of an opal codon between nsP3 and nsP4 is related to mosquito infectivity.

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