Stop Codon Readthrough Research Articles

RiboTools: a Galaxy toolbox for qualitative ribosome profiling analysis.

Ribosome profiling provides genome-wide information about translational regulation. However, there is currently no standard tool for the qualitative analysis of Ribo-seq data. We present here RiboTools, a Galaxy toolbox for the analysis of ribosome profiling (Ribo-seq) data. It can be used to detect translational ambiguities, stop codon readthrough events and codon occupancy. It provides a large number of plots for the visualisation of these events.

Comparison of mutation profiles in the Duchenne muscular dystrophy gene among populations: implications for potential molecular therapies.

Novel therapeutic approaches are emerging to restore dystrophin function in Duchenne Muscular Dystrophy (DMD), a severe neuromuscular disease characterized by progressive muscle wasting and weakness. Some of the molecular therapies, such as exon skipping, stop codon read-through and internal ribosome entry site-mediated translation rely on the type and location of mutations. Hence, their potential applicability worldwide depends on mutation frequencies within populations. In view of this, we compared the mutation profiles of the populations represented in the DMD Leiden Open-source Variation Database with original data from Mexican patients (n = 162) with clinical diagnosis of the disease. Our data confirm that applicability of exon 51 is high in most populations, but also show that differences in theoretical applicability of exon skipping may exist among populations; Mexico has the highest frequency of potential candidates for the skipping of exons 44 and 46, which is different from other populations (p < 0.001). To our knowledge, this is the first comprehensive comparison of theoretical applicability of exon skipping targets among specific populations.

Mechanisms of mRNA frame maintenance and its subversion during translation of the genetic code.

Important viral and cellular gene products are regulated by stop codon readthrough and mRNA frameshifting, processes whereby the ribosome detours from the reading frame defined by three nucleotide codons after initiation of translation. In the last few years, rapid progress has been made in mechanistically characterizing both processes and also revealing that trans-acting factors play important regulatory roles in frameshifting. Here, we review recent biophysical studies that bring new molecular insights to stop codon readthrough and frameshifting. Lastly, we consider whether there may be common mechanistic themes in -1 and +1 frameshifting based on recent X-ray crystal structures of +1 frameshift-prone tRNAs bound to the ribosome.

Nonsense codon suppression in fission yeast due to mutations of tRNA(Ser.11) and translation release factor Sup35 (eRF3).

In the fission yeast Schizosaccharomyces pombe, sup9 mutations can suppress the termination of translation at nonsense (stop) codons. We localized sup9 physically to the spctrnaser.11 locus and confirmed that one allele (sup9-UGA) alters the anticodon of a serine tRNA. We also found that another purported allele is not allelic. Instead, strains with that suppressor (renamed sup35-F592S) have a single base pair substitution (T1775C) that introduces an amino acid substitution in the Sup35 protein (Sup35-F592S). Reduced functionality of Sup35 (eRF3), the ubiquitous guanine nucleotide-responsive translation release factor of eukaryotes, increases read-through of stop codons. Tetrad dissection revealed that suppression is tightly linked to (inseparable from) the sup35-F592S mutation and that there are no additional extragenic modifiers. The Mendelian inheritance indicates that the Sup35-F592S protein does not adopt an infectious amyloid state ([PSI (+)] prion) to affect suppression, consistent with recent evidence that fission yeast Sup35 does not form prions. We also report that sup9-UGA and sup35-F592S exhibit different strengths of suppression for opal stop codons of ade6-M26 and ade6-M375. We discuss possible mechanisms for the variation in suppressibility exhibited by the two alleles.

Efficient multisite unnatural amino acid incorporation in mammalian cells via optimized pyrrolysyl tRNA synthetase/tRNA expression and engineered eRF1.

The efficient, site-specific introduction of unnatural amino acids into proteins in mammalian cells is an outstanding challenge in realizing the potential of genetic code expansion approaches. Addressing this challenge will allow the synthesis of modified recombinant proteins and augment emerging strategies that introduce new chemical functionalities into proteins to control and image their function with high spatial and temporal precision in cells. The efficiency of unnatural amino acid incorporation in response to the amber stop codon (UAG) in mammalian cells is commonly considered to be low. Here we demonstrate that tRNA levels can be limiting for unnatural amino acid incorporation efficiency, and we develop an optimized pyrrolysyl-tRNA synthetase/tRNACUA expression system, with optimized tRNA expression for mammalian cells. In addition, we engineer eRF1, that normally terminates translation on all three stop codons, to provide a substantial increase in unnatural amino acid incorporation in response to the UAG codon without increasing readthrough of other stop codons. By combining the optimized pyrrolysyl-tRNA synthetase/tRNACUA expression system and an engineered eRF1, we increase the yield of protein bearing unnatural amino acids at a single site 17- to 20-fold. Using the optimized system, we produce proteins containing unnatural amino acids with comparable yields to a protein produced from a gene that does not contain a UAG stop codon. Moreover, the optimized system increases the yield of protein, incorporating an unnatural amino acid at three sites, from unmeasurably low levels up to 43% of a no amber stop control. Our approach may enable the efficient production of site-specifically modified therapeutic proteins, and the quantitative replacement of targeted cellular proteins with versions bearing unnatural amino acids that allow imaging or synthetic regulation of protein function.

Ribosomal readthrough at a short UGA stop codon context triggers dual localization of metabolic enzymes in Fungi and animals.

Translation of mRNA into a polypeptide chain is a highly accurate process. Many prokaryotic and eukaryotic viruses, however, use leaky termination of translation to optimize their coding capacity. Although growing evidence indicates the occurrence of ribosomal readthrough also in higher organisms, a biological function for the resulting extended proteins has been elucidated only in very few cases. Here, we report that in human cells programmed stop codon readthrough is used to generate peroxisomal isoforms of cytosolic enzymes. We could show for NAD-dependent lactate dehydrogenase B (LDHB) and NAD-dependent malate dehydrogenase 1 (MDH1) that translational readthrough results in C-terminally extended protein variants containing a peroxisomal targeting signal 1 (PTS1). Efficient readthrough occurs at a short sequence motif consisting of a UGA termination codon followed by the dinucleotide CU. Leaky termination at this stop codon context was observed in fungi and mammals. Comparative genome analysis allowed us to identify further readthrough-derived peroxisomal isoforms of metabolic enzymes in diverse model organisms. Overall, our study highlights that a defined stop codon context can trigger efficient ribosomal readthrough to generate dually targeted protein isoforms. We speculate that beyond peroxisomal targeting stop codon readthrough may have also other important biological functions, which remain to be elucidated.

Ataluren treatment of patients with nonsense mutation dystrophinopathy.

Introduction: Dystrophinopathy is a rare, severe muscle disorder, and nonsense mutations are found in 13% of cases. Ataluren was developed to enable ribosomal readthrough of premature stop codons in nonsense mutation (nm) genetic disorders. Methods: Randomized, double-blind, placebo-controlled study; males ≥5 years with nm-dystrophinopathy received study drug orally 3 times daily, ataluren 10, 10, 20 mg/kg (N = 57); ataluren 20, 20, 40 mg/kg (N = 60); or placebo (N = 57) for 48 weeks. The primary endpoint was change in 6-Minute Walk Distance (6MWD) at Week 48. Results: Ataluren was generally well tolerated. The primary endpoint favored ataluren 10, 10, 20 mg/kg versus placebo; the week 48 6MWD Δ = 31.3 meters, post hoc P = 0.056. Secondary endpoints (timed function tests) showed meaningful differences between ataluren 10, 10, 20 mg/kg, and placebo. Conclusions: As the first investigational new drug targeting the underlying cause of nm-dystrophinopathy, ataluren offers promise as a treatment for this orphan genetic disorder with high unmet medical need. Muscle Nerve 50: 477–487, 2014

G.P.106: Internal consistency and robustness of clinical efficacy data from the phase 2b study of ataluren (PTC124®) in nonsense mutation Duchenne muscular dystrophy

Ataluren is an oral, investigational drug designed to promote ribosomal readthrough of premature stop codons in mRNA, leading to production of full-length, functional protein. In a Phase 2b, 48-week study in nonsense mutation Duchenne muscular dystrophy (nmDMD), ataluren 40 mg/kg/day was associated with slowing of ambulatory decline (measured by 6-min walk distance [6MWD]) vs placebo. Positive trends were also seen in timed function tests of stair-climbing, stair-descending, and walking/running 10 m. Treatment differences vs placebo were larger in an ambulatory decline phase subgroup (7–16 yrs old, on corticosteroids, baseline 6MWD ¡Ý150 m but %-predicted 6MWD ¡Ü80%). Internal consistency and robustness of physical function outcomes were evaluated by statistical methods. Endpoint analyses were repeated 1000 times (Monte Carlo simulation), each time with 10% of patients in each arm excluded at random. Separately, endpoint analyses were performed after excluding one or more extreme results (best/worst exclusion) from each arm. These methods were applied to the overall study population and ambulatory decline phase subgroup. Monte Carlo simulation demonstrated that treatment differences in model-estimated mean change in 6MWD of ∼ 30 m in the overall study population and ∼45 m in the ambulatory decline phase subgroup, favoring ataluren vs placebo, were not driven by a small number of patients. Best/worst exclusion analysis confirmed these treatment differences were not distorted by outliers. Timed function test outcomes were consistent with 6MWD findings. Ambulatory decline in patients with nmDMD could potentially be delayed or stabilized with disease-modifying treatment. These analyses document the internal consistency and give evidence of robustness of the Phase 2b efficacy data for ataluren in nmDMD, confirming observations of relative stabilization of ambulatory measures of disease progression over 48 weeks with ataluren. Ataluren is an oral, investigational drug designed to promote ribosomal readthrough of premature stop codons in mRNA, leading to production of full-length, functional protein. In a Phase 2b, 48-week study in nonsense mutation Duchenne muscular dystrophy (nmDMD), ataluren 40 mg/kg/day was associated with slowing of ambulatory decline (measured by 6-min walk distance [6MWD]) vs placebo. Positive trends were also seen in timed function tests of stair-climbing, stair-descending, and walking/running 10 m. Treatment differences vs placebo were larger in an ambulatory decline phase subgroup (7–16 yrs old, on corticosteroids, baseline 6MWD ¡Ý150 m but %-predicted 6MWD ¡Ü80%). Internal consistency and robustness of physical function outcomes were evaluated by statistical methods. Endpoint analyses were repeated 1000 times (Monte Carlo simulation), each time with 10% of patients in each arm excluded at random. Separately, endpoint analyses were performed after excluding one or more extreme results (best/worst exclusion) from each arm. These methods were applied to the overall study population and ambulatory decline phase subgroup. Monte Carlo simulation demonstrated that treatment differences in model-estimated mean change in 6MWD of ∼ 30 m in the overall study population and ∼45 m in the ambulatory decline phase subgroup, favoring ataluren vs placebo, were not driven by a small number of patients. Best/worst exclusion analysis confirmed these treatment differences were not distorted by outliers. Timed function test outcomes were consistent with 6MWD findings. Ambulatory decline in patients with nmDMD could potentially be delayed or stabilized with disease-modifying treatment. These analyses document the internal consistency and give evidence of robustness of the Phase 2b efficacy data for ataluren in nmDMD, confirming observations of relative stabilization of ambulatory measures of disease progression over 48 weeks with ataluren.

New insights into the incorporation of natural suppressor tRNAs at stop codons in Saccharomyces cerevisiae.

Stop codon readthrough may be promoted by the nucleotide environment or drugs. In such cases, ribosomes incorporate a natural suppressor tRNA at the stop codon, leading to the continuation of translation in the same reading frame until the next stop codon and resulting in the expression of a protein with a new potential function. However, the identity of the natural suppressor tRNAs involved in stop codon readthrough remains unclear, precluding identification of the amino acids incorporated at the stop position. We established an in vivo reporter system for identifying the amino acids incorporated at the stop codon, by mass spectrometry in the yeast Saccharomyces cerevisiae. We found that glutamine, tyrosine and lysine were inserted at UAA and UAG codons, whereas tryptophan, cysteine and arginine were inserted at UGA codon. The 5′ nucleotide context of the stop codon had no impact on the identity or proportion of amino acids incorporated by readthrough. We also found that two different glutamine tRNAGln were used to insert glutamine at UAA and UAG codons. This work constitutes the first systematic analysis of the amino acids incorporated at stop codons, providing important new insights into the decoding rules used by the ribosome to read the genetic code.

Evidence of efficient stop codon readthrough in four mammalian genes.

Stop codon readthrough is used extensively by viruses to expand their gene expression. Until recent discoveries in Drosophila, only a very limited number of readthrough cases in chromosomal genes had been reported. Analysis of conserved protein coding signatures that extend beyond annotated stop codons identified potential stop codon readthrough of four mammalian genes. Here we use a modified targeted bioinformatic approach to identify a further three mammalian readthrough candidates. All seven genes were tested experimentally using reporter constructs transfected into HEK-293T cells. Four displayed efficient stop codon readthrough, and these have UGA immediately followed by CUAG. Comparative genomic analysis revealed that in the four readthrough candidates containing UGA-CUAG, this motif is conserved not only in mammals but throughout vertebrates with the first six of the seven nucleotides being universally conserved. The importance of the CUAG motif was confirmed using a systematic mutagenesis approach. One gene, OPRL1, encoding an opiate receptor, displayed extremely efficient levels of readthrough (∼31%) in HEK-293T cells. Signals both 5′ and 3′ of the OPRL1 stop codon contribute to this high level of readthrough. The sequence UGA-CUA alone can support 1.5% readthrough, underlying its importance.

Modulation of stop codon read-through efficiency and its effect on the replication of murine leukemia virus.

ABSTRACTTranslational readthrough—suppression of termination at a stop codon—is exploited in the replication cycles of several viruses and represents a potential target for antiviral intervention. In the gammaretroviruses, typified by Moloney murine leukemia virus (MuLV), gag and pol are in the same reading frame, separated by a UAG stop codon, and termination codon readthrough is required for expression of the viral Gag-Pol fusion protein. Here, we investigated the effect on MuLV replication of modulating readthrough efficiency. We began by manipulating the readthrough signal in the context of an infectious viral clone to generate a series of MuLV variants in which readthrough was stimulated or reduced. In carefully controlled infectivity assays, it was found that reducing the MuLV readthrough efficiency only 4-fold led to a marked defect and that a 10-fold reduction essentially abolished replication. However, up to an ∼8.5-fold stimulation of readthrough (up to 60% readthrough) was well tolerated by the virus. These high levels of readthrough were achieved using a two-plasmid system, with Gag and Gag-Pol expressed from separate infectious clones. We also modulated readthrough by silencing expression of eukaryotic release factors 1 and 3 (eRF1 and eRF3) or by introducing aminoglycosides into the cells. The data obtained indicate that gammaretroviruses tolerate a substantial excess of viral Gag-Pol synthesis but are very sensitive to a reduction in levels of this polyprotein. Thus, as is also the case for ribosomal frameshifting, antiviral therapies targeting readthrough with inhibitory agents are likely to be the most beneficial.IMPORTANCE Many pathogenic RNA viruses and retroviruses use ribosomal frameshifting or stop codon readthrough to regulate expression of their replicase enzymes. These translational “recoding” processes are potential targets for antiviral intervention, but we have only a limited understanding of the consequences to virus replication of modulating the efficiency of recoding, particularly for those viruses employing readthrough. In this paper, we describe the first systematic analysis of the effect of increasing or decreasing readthrough efficiency on virus replication using the gammaretrovirus MuLV as a model system. We find unexpectedly that MuLV replication is only slightly inhibited by substantial increases in readthrough frequency, but as with other viruses that use recoding strategies, replication is quite sensitive to even modest reductions. These studies provide insights into both the readthrough process and MuLV replication and have implications for the selection of antivirals against gammaretroviruses.

Genome-wide Translational Changes Induced by the Prion [PSI+

Prions are infectious proteins that can adopt a structural conformation that is then propagated among other molecules of the same protein. [PSI(+)] is an aggregated conformation of the translational release factor eRF3. [PSI(+)] modifies cellular fitness, inducing various phenotypes depending on genetic background. However, the genes displaying [PSI(+)]-controlled expression remain unknown. We used ribosome profiling in isogenic [PSI(+)] and [psi(-)] strains to identify the changes induced by [PSI(+)]. We found 100 genes with stop codon readthrough events and showed that many stress-response genes were repressed in the presence of [PSI(+)]. Surprisingly, [PSI(+)] was also found to affect reading frame selection independently of its effect on translation termination efficiency. These results indicate that [PSI(+)] has a broader impact than initially anticipated, providing explanations for the phenotypic differences between [psi(-)] and [PSI(+)] strains.

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

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

Therapeutics based on stop codon readthrough.

Nonsense suppression therapy encompasses approaches aimed at suppressing translation termination at in-frame premature termination codons (PTCs, also known as nonsense mutations) to restore deficient protein function. In this review, we examine the current status of PTC suppression as a therapy for genetic diseases caused by nonsense mutations. We discuss what is currently known about the mechanism of PTC suppression as well as therapeutic approaches under development to suppress PTCs. The approaches considered include readthrough drugs, suppressor tRNAs, PTC pseudouridylation, and inhibition of nonsense-mediated mRNA decay. We also discuss the barriers that currently limit the clinical application of nonsense suppression therapy and suggest how some of these difficulties may be overcome. Finally, we consider how PTC suppression may play a role in the clinical treatment of genetic diseases caused by nonsense mutations.

The ribosome‐associated Hsp40 chaperone Zuo1 antagonizes prion formation in yeast (752.11)

Prions are self‐propagating infectious protein conformations. In mammals prions underlie the transmissible spongiform encephalopathies, a set of invariably fatal neurodegenerative disorders associated with misfolding of the mammalian prion protein, PrP. In recent years increasing numbers of fungal proteins capable of infectiously propagating altered conformations have been discovered. Fungal prions are typically not lethal and debate continues as to whether they may have evolutionarily conserved physiological roles rather than representing disease states. To gain insight into the mechanisms by which cells regulate prion formation, propagation and toxicity, we have examined the role of the ribosome‐associated complex (RAC) chaperones in the formation and toxicity of the [PSI+] prion in yeast. The [PSI+] prion results from a self‐propagating aggregated form of the essential translation termination factor Sup35, and is manifested phenotypically as elevated levels of nonsense suppression. We find that yeast cells lacking the Hsp40 chaperone Zuo1 exhibit higher frequencies of spontaneous and induced [PSI+] formation, consistent with a role for the RAC in chaperoning nascent Sup35 to prevent misfolding of the N‐terminal prion domain as it emerges from the ribosome. Notably, formation of [PSI+] in cells lacking Zuo1 is associated with a reduction in fitness. As both ZUO1 deletion and the [PSI+] prion can independently enhance readthrough of stop codons, toxicity may be due to the elevated levels of nonsense suppression in Δzuo1 [PSI+] strains. Studies are currently underway to understand the mechanisms by which deletion of Zuo1 enhances both the formation and toxicity of the [PSI+] prion.Grant Funding Source: Supported by Research Corporation for Science Advancement

Annotating Viral Genomes - A Cannon is Needed to Kill Mosquitoes

The majority of viruses have a small genome. However, these small genomes often have complex gene features with transcriptional and translational exceptions, for instance, gene overlapping, alternative splicing, RNA editing, ribosomal slippage and stop codon read-through. These complex features and exceptions increase gene density and improve the gene coding efficiency of viral genomes. They also pose immense challenges to gene prediction algorithms. Most gene prediction programs for eukaryotic and prokaryotic genomes cannot detect or predict these exceptions correctly. It is critical to predict these complex features and exceptions with high precision and accuracy in order to interpret viral genomic data correctly. This paper describes the most commonly used programs for viral gene predictions, focusing on the ab initio and similarity-based gene prediction programs, including GeneMarkS, ZCURVE_V, FgenesV, Phylo-HMM, MLOGD, GATU, VirGen, FLAN, VIGOR and others. Viral genome complex features and the basic algorithms of the gene prediction programs are introduced briefly, with identification of advantages and disadvantages, followed by a list of application scopes and specific features. Gene prediction programs for bacteriophages and viral meta-genomic sequences are reviewed separately. The last section of this review presents the future directions and challenges for viral gene prediction program development. Keywords: Mature peptide prediction, viral gene complex feature, viral gene prediction, viral genome annotation, VIGOR.

Cryptic MHC class I-binding peptides are revealed by aminoglycoside-induced stop codon read-through into the 3′ UTR

Aminoglycosides have been proposed as therapies for genetic disorders caused by nonsense mutations, because of their capacity to enhance translational read-through of premature termination codons (PTCs), thereby permitting expression of functional full-length protein. However, a potential consequence of this strategy is the development of an autoimmune response to HLA-presented epitopes encoded downstream of the PTC or other stop codons. Using a recombinant virus-expression system in tissue culture and in mice, we demonstrate that gentamicin can induce expression and MHC class I presentation of a model epitope encoded downstream of a PTC at levels sufficient to activate CD8(+) T cells. The degree of read-through-derived peptide presentation varies with the sequence of the stop codon and +1 nucleotide. Additionally, we applied a mass spectrometry exploration of the HLA class I peptide repertoire of gentamicin-treated cells and identified multiple peptides derived from read-through of conventional stop codons. These results substantiate the possibility of self-reactivity to cryptic epitopes revealed by stop codon read-through therapies and potentially other therapeutic approaches involving compounds that alter translational fidelity.

S1 Development of a confirmatory phase 3, multicentre, randomized, double-blind, placebo-controlled study of ataluren in patients with nonsense mutation Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a rare, X-linked disorder that causes severe, progressive muscle loss and early death. In ∼13% of patients, the disease is caused by a nonsense mutation (nm) in the gene for dystrophin. Ataluren is an orally delivered, investigational drug designed to promote ribosomal readthrough of premature stop codons in mRNA, leading to production of full-length, functional protein. A confirmatory Phase 3, multicenter, randomized, double-blind, placebo-controlled study has been designed to assess the efficacy and safety of ataluren 10, 10, 20 mg/kg tid in patients with nmDMD. The study design reflects lessons learned from prior studies and targets a study population to best demonstrate the treatment effect over 48 weeks. Key study entry criteria require that patients are male with a nonsense mutation in the dystrophin gene, between the ages of 7 and 16 years, receiving a stable dose of corticosteroids, able to walk ⩾150 m during the screening 6-min walk test (6MWT), and have a screening 6MWT below the protocol-specified threshold for %-predicted 6MWD. These criteria were selected based on the results of a retrospective subgroup analysis of patients in the Phase 2b trial of ataluren in nmDMD meeting these criteria, in which the difference between the 10, 10, 20 mg/kg dose of ataluren ( n = 30) vs placebo ( n = 31) in mean change in 6MWD over 48 weeks was ∼50 m. In the planned study, 220 patients will be randomized in a 1:1 ratio to either placebo or ataluren. The primary outcome measure is the 6MWT. Secondary measures include: timed function tests, quality of life, North Star Ambulatory Assessment, patient/parent-reported activities of daily living, safety, compliance to study drug, and ataluren exposure of ataluren in blood. This study will be the largest clinical trial of an investigational drug in DMD and is designed to confirm the treatment effect of ataluren seen in the Phase 2b ataluren trial.

Hydroxylation of the eukaryotic ribosomal decoding center affects translational accuracy

The mechanisms by which gene expression is regulated by oxygen are of considerable interest from basic science and therapeutic perspectives. Using mass spectrometric analyses of Saccharomyces cerevisiae ribosomes, we found that the amino acid residue in closest proximity to the decoding center, Pro-64 of the 40S subunit ribosomal protein Rps23p (RPS23 Pro-62 in humans) undergoes posttranslational hydroxylation. We identify RPS23 hydroxylases as a highly conserved eukaryotic subfamily of Fe(II) and 2-oxoglutarate dependent oxygenases; their catalytic domain is closely related to transcription factor prolyl trans-4-hydroxylases that act as oxygen sensors in the hypoxic response in animals. The RPS23 hydroxylases in S. cerevisiae (Tpa1p), Schizosaccharomyces pombe and green algae catalyze an unprecedented dihydroxylation modification. This observation contrasts with higher eukaryotes, where RPS23 is monohydroxylated; the human Tpa1p homolog OGFOD1 catalyzes prolyl trans-3-hydroxylation. TPA1 deletion modulates termination efficiency up to ∼10-fold, including of pathophysiologically relevant sequences; we reveal Rps23p hydroxylation as its molecular basis. In contrast to most previously characterized accuracy modulators, including antibiotics and the prion state of the S. cerevisiae translation termination factor eRF3, Rps23p hydroxylation can either increase or decrease translational accuracy in a stop codon context-dependent manner. We identify conditions where Rps23p hydroxylation status determines viability as a consequence of nonsense codon suppression. The results reveal a direct link between oxygenase catalysis and the regulation of gene expression at the translational level. They will also aid in the development of small molecules altering translational accuracy for the treatment of genetic diseases linked to nonsense mutations.

Toward a rationale for the PTC124 (Ataluren) promoted readthrough of premature stop codons: a computational approach and GFP-reporter cell-based assay.

The presence in the mRNA of premature stop codons (PTCs) results in protein truncation responsible for several inherited (genetic) diseases. A well-known example of these diseases is cystic fibrosis (CF), where approximately 10% (worldwide) of patients have nonsense mutations in the CF transmembrane regulator (CFTR) gene. PTC124 (3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)-benzoic acid), also known as Ataluren, is a small molecule that has been suggested to allow PTC readthrough even though its target has yet to be identified. In the lack of a general consensus about its mechanism of action, we experimentally tested the ability of PTC124 to promote the readthrough of premature termination codons by using a new reporter. The reporter vector was based on a plasmid harboring the H2B histone coding sequence fused in frame with the green fluorescent protein (GFP) cDNA, and a TGA stop codon was introduced in the H2B-GFP gene by site-directed mutagenesis. Additionally, an unprecedented computational study on the putative supramolecular interaction between PTC124 and an 11-codon (33-nucleotides) sequence corresponding to a CFTR mRNA fragment containing a central UGA nonsense mutation showed a specific interaction between PTC124 and the UGA codon. Altogether, the H2B-GFP-opal based assay and the molecular dynamics (MD) simulation support the hypothesis that PTC124 is able to promote the specific readthrough of internal TGA premature stop codons.