Translational readthrough at nonsense mutations in the HSF1 gene of Saccharomyces cerevisme

ABSTRACT Translational readthrough of nonsense codons is seen not only in organisms possessing one or more tRNA suppressors but also in strains lacking suppressors. Amber suppressor tRNAs have been reported to suppress only amber nonsense mutations, unlike ochre suppressors, which can suppress both amber and ochre mutations, essentially due to wobble base pairing. In an Escherichia coli strain carrying the lacZU118 episome (an ochre mutation in the lacZ gene) and harboring the supE44 allele, suppression of the ochre mutation was observed after 7 days of incubation. The presence of the supE44 lesion in the relevant strains was confirmed by sequencing, and it was found to be in the duplicate copy of the glnV tRNA gene, glnX. To investigate this further, an in vivo luciferase assay developed by D. W. Schultz and M. Yarus (J. Bacteriol. 172:595-602, 1990) was employed to evaluate the efficiency of suppression of amber (UAG), ochre (UAA), and opal (UGA) mutations by supE44. We have shown here that supE44 suppresses ochre as well as opal nonsense mutations, with comparable efficiencies. The readthrough of nonsense mutations in a wild-type E. coli strain was much lower than that in a supE44 strain when measured by the luciferase assay. Increased suppression of nonsense mutations, especially ochre and opal, by supE44 was found to be growth phase dependent, as this phenomenon was only observed in stationary phase and not in logarithmic phase. These results have implications for the decoding accuracy of the translational machinery, particularly in stationary growth phase.

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Nonsense suppression of the major rhodopsin gene of Drosophila.

Genetics ◽

10.1093/genetics/130.3.585 ◽

1992 ◽

Vol 130 (3) ◽

pp. 585-595 ◽

Cited By ~ 1

Author(s):

T Washburn ◽

J E O'Tousa

Keyword(s):

Deletion Mutant ◽

Trna Gene ◽

Site Directed Mutagenesis ◽

P Element ◽

Nonsense Suppression ◽

Nonsense Mutations ◽

Rhodopsin Gene ◽

Translational Readthrough ◽

Mutant Strains ◽

Nonsense Suppressors

Abstract We placed UAA, UAG and UGA nonsense mutations at two leucine codons, Leu205 and Leu309, in Drosophila's major rhodopsin gene, ninaE, by site-directed mutagenesis, and then created the corresponding mutants by P element-mediated transformation of a ninaE deficiency strain. In the absence of a genetic suppressor, flies harboring any of the nonsense mutations at the 309 site, but not the 205 site, show increased rhodopsin activity. Additionally, all flies with nonsense mutations at either site have better rhabdomere structure than does the ninaE deficiency strain. Construction and analysis of a 3'-deletion mutant of ninaE indicates that translational readthrough accounts for the extra photoreceptor activity of the ninaE309 alleles and that truncated opsins are responsible for the improved rhabdomere structure. The presence of leucine-inserting tRNA nonsense suppressors DtLa Su+ and DtLb Su+ in the mutant strains produced a small increase (less than 0.04%) in functional rhodopsin. The opal (UGA) suppressor derived from the DtLa tRNA gene is more efficient than the amber (UAG) or opal suppressor derived from the DtLb gene, and both DtLa and DtLb derived suppressors are more efficient at site 205 than 309.

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CFTR mRNAs with nonsense codons are degraded by the SMG6-mediated endonucleolytic pathway

10.21203/rs.3.rs-800274/v1 ◽

2021 ◽

Author(s):

Edward Sanderlin ◽

Melissa Keenan ◽

Martin Mense ◽

Alexey Revenko ◽

Brett Monia ◽

...

Keyword(s):

Cystic Fibrosis ◽

Exon Junction Complex ◽

Transmembrane Conductance Regulator ◽

Loss Of Function ◽

Transmembrane Conductance ◽

Nonsense Mutations ◽

Nonsense Mediated Decay ◽

Nonsense Codon ◽

Translational Readthrough ◽

Nonsense Codons

Abstract Cystic fibrosis is caused by loss of function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene resulting in severe lung disease. Nearly 10% of cystic fibrosis patients have at least one CFTR allele with a nonsense mutation that generates a nonsense codon in the mRNA. Nonsense mutations can result in significant reduction of gene expression partially due to rapid mRNA degradation through the nonsense-mediated decay (NMD) pathway. It has not been thoroughly investigated which branch of the NMD pathway governs the decay of CFTR mRNAs containing nonsense codons. Here we utilized antisense oligonucleotides targeting NMD factors to evaluate the regulation of nonsense codon-containing CFTR mRNAs by the NMD pathway. Interestingly, we found that CFTR mRNAs with G542X, R1162X, and W1282X nonsense codons require UPF2, UPF3, and exon junction complex proteins for NMD, whereas CFTR mRNAs with the Y122X nonsense codon do not. Furthermore, we demonstrated that all evaluated CFTR mRNAs harboring nonsense codons were degraded by the SMG6-mediated endonucleolytic pathway rather than the SMG5/SMG7-mediated exonucleolytic pathway. Finally, we found that stabilization of CFTR mRNAs by NMD inhibition alone improved functional W1282X protein production, and improved the efficiency of aminoglycoside translational readthrough of CFTR-Y122X, -G542X, and -R1162X mRNAs.

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Targeting Nonsense: Optimization of 1,2,4-Oxadiazole TRIDs to Rescue CFTR Expression and Functionality in Cystic Fibrosis Cell Model Systems

International Journal of Molecular Sciences ◽

10.3390/ijms21176420 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6420

Author(s):

Ivana Pibiri ◽

Raffaella Melfi ◽

Marco Tutone ◽

Aldo Di Leonardo ◽

Andrea Pace ◽

...

Keyword(s):

Cystic Fibrosis ◽

Fluorescent Protein ◽

Cell Model ◽

Yellow Fluorescent Protein ◽

Premature Termination Codon ◽

Severe Form ◽

Model Systems ◽

Functional Protein ◽

Nonsense Mutations ◽

Translational Readthrough

Cystic fibrosis (CF) patients develop a severe form of the disease when the cystic fibrosis transmembrane conductance regulator (CFTR) gene is affected by nonsense mutations. Nonsense mutations are responsible for the presence of a premature termination codon (PTC) in the mRNA, creating a lack of functional protein. In this context, translational readthrough-inducing drugs (TRIDs) represent a promising approach to correct the basic defect caused by PTCs. By using computational optimization and biological screening, we identified three new small molecules showing high readthrough activity. The activity of these compounds has been verified by evaluating CFTR expression and functionality after treatment with the selected molecules in cells expressing nonsense–CFTR–mRNA. Additionally, the channel functionality was measured by the halide sensitive yellow fluorescent protein (YFP) quenching assay. All three of the new TRIDs displayed high readthrough activity and low toxicity and can be considered for further evaluation as a therapeutic approach toward the second major cause of CF.

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Strategies against Nonsense: Oxadiazoles as Translational Readthrough-Inducing Drugs (TRIDs)

International Journal of Molecular Sciences ◽

10.3390/ijms20133329 ◽

2019 ◽

Vol 20 (13) ◽

pp. 3329 ◽

Cited By ~ 10

Author(s):

Ambra Campofelice ◽

Laura Lentini ◽

Aldo Di Leonardo ◽

Raffaella Melfi ◽

Marco Tutone ◽

...

Keyword(s):

Small Molecules ◽

Genetic Diseases ◽

Nonsense Mutations ◽

New Class ◽

Premature Termination Codons ◽

Translational Readthrough ◽

Action Strategies ◽

Oxadiazole Derivatives ◽

Cftr Protein ◽

Cystic Fibrosis Transmembrane

This review focuses on the use of oxadiazoles as translational readthrough-inducing drugs (TRIDs) to rescue the functional full-length protein expression in mendelian genetic diseases caused by nonsense mutations. These mutations in specific genes generate premature termination codons (PTCs) responsible for the translation of truncated proteins. After a brief introduction on nonsense mutations and their pathological effects, the features of various classes of TRIDs will be described discussing differences or similarities in their mechanisms of action. Strategies to correct the PTCs will be presented, particularly focusing on a new class of Ataluren-like oxadiazole derivatives in comparison to aminoglycosides. Additionally, recent results on the efficiency of new candidate TRIDs in restoring the production of the cystic fibrosis transmembrane regulator (CFTR) protein will be presented. Finally, a prospectus on complementary strategies to enhance the effect of TRIDs will be illustrated together with a conclusive paragraph about perspectives, opportunities, and caveats in developing small molecules as TRIDs.

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Molecular Insights into Determinants of Translational Readthrough and Implications for Nonsense Suppression Approaches

International Journal of Molecular Sciences ◽

10.3390/ijms21249449 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9449

Author(s):

Silvia Lombardi ◽

Maria Francesca Testa ◽

Mirko Pinotti ◽

Alessio Branchini

Keyword(s):

Therapeutic Strategy ◽

Low Frequency ◽

External Factors ◽

Full Length ◽

Nonsense Suppression ◽

Coagulation Disorders ◽

Nonsense Mutations ◽

Premature Termination Codons ◽

Translational Readthrough ◽

Molecular Determinants

The fidelity of protein synthesis, a process shaped by several mechanisms involving specialized ribosome regions and external factors, ensures the precise reading of sense and stop codons. However, premature termination codons (PTCs) arising from mutations may, at low frequency, be misrecognized and result in PTC suppression, named ribosome readthrough, with production of full-length proteins through the insertion of a subset of amino acids. Since some drugs have been identified as readthrough inducers, this fidelity drawback has been explored as a therapeutic approach in several models of human diseases caused by nonsense mutations. Here, we focus on the mechanisms driving translation in normal and aberrant conditions, the potential fates of mRNA in the presence of a PTC, as well as on the results obtained in the research of efficient readthrough-inducing compounds. In particular, we describe the molecular determinants shaping the outcome of readthrough, namely the nucleotide and protein context, with the latter being pivotal to produce functional full-length proteins. Through the interpretation of experimental and mechanistic findings, mainly obtained in lysosomal and coagulation disorders, we also propose a scenario of potential readthrough-favorable features to achieve relevant rescue profiles, representing the main issue for the potential translatability of readthrough as a therapeutic strategy.

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A small molecule that induces translational readthrough of CFTR nonsense mutations by eRF1 depletion

Nature Communications ◽

10.1038/s41467-021-24575-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jyoti Sharma ◽

Ming Du ◽

Eric Wong ◽

Venkateshwar Mutyam ◽

Yao Li ◽

...

Keyword(s):

Protein Function ◽

Translation Termination ◽

Nonsense Suppression ◽

Nonsense Mutations ◽

Bronchial Epithelial ◽

Premature Termination Codons ◽

Translational Readthrough ◽

Nonsense Mediated Mrna Decay ◽

Promising Treatment ◽

And Function

AbstractPremature termination codons (PTCs) prevent translation of a full-length protein and trigger nonsense-mediated mRNA decay (NMD). Nonsense suppression (also termed readthrough) therapy restores protein function by selectively suppressing translation termination at PTCs. Poor efficacy of current readthrough agents prompted us to search for better compounds. An NMD-sensitive NanoLuc readthrough reporter was used to screen 771,345 compounds. Among the 180 compounds identified with readthrough activity, SRI-37240 and its more potent derivative SRI-41315, induce a prolonged pause at stop codons and suppress PTCs associated with cystic fibrosis in immortalized and primary human bronchial epithelial cells, restoring CFTR expression and function. SRI-41315 suppresses PTCs by reducing the abundance of the termination factor eRF1. SRI-41315 also potentiates aminoglycoside-mediated readthrough, leading to synergistic increases in CFTR activity. Combining readthrough agents that target distinct components of the translation machinery is a promising treatment strategy for diseases caused by PTCs.

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Translational readthrough induction of pathogenic nonsense mutations

European Journal of Medical Genetics ◽

10.1016/j.ejmg.2006.04.003 ◽

2006 ◽

Vol 49 (6) ◽

pp. 445-450 ◽

Cited By ~ 43

Author(s):

Richard Kellermayer

Keyword(s):

Nonsense Mutations ◽

Translational Readthrough

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Translational readthrough as a potential therapeutic for AIPL1-associated Leber Congenital Amaurosis in a patient-derived iPSC-retinal organoid model

10.1101/2021.12.17.473147 ◽

2021 ◽

Author(s):

Amy Leung ◽

Almudena Sacristan-Reviriego ◽

Pedro Perdigao ◽

Hali Sai ◽

Michalis Georgiou ◽

...

Keyword(s):

Pluripotent Stem Cells ◽

Molecular Mechanisms ◽

Photoreceptor Cells ◽

Nonsense Mutations ◽

Leber Congenital Amaurosis ◽

Progressive Degeneration ◽

In Vitro Investigation ◽

Translational Readthrough ◽

Induced Pluripotent

Leber Congenital Amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterised by severe sight impairment in infancy and rapidly progressive degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cGMP phosphodiesterase (PDE6) compared to control or CRISPR corrected organoids. Moreover, increased levels of cGMP were detected. The translational readthrough inducing drug (TRID) PTC124 was investigated as a potential therapeutic. LCA4 retinal organoids exhibited rescue of AIPL1 and PDE6; however, the level of full-length, functional AIPL1 induced through PTC124 treatment was insufficient to reduce cGMP levels and fully rescue the LCA4 phenotype. LCA4 retinal organoids are a valuable platform for the in vitro investigation of the molecular mechanisms that drive photoreceptor loss and for the evaluation of novel therapeutics.

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