scholarly journals Suppression of aminoglycoside-induced premature termination codon readthrough by the TRP channel inhibitor AC1903

2021 ◽  
Author(s):  
Alireza Baradaran-Heravi ◽  
Claudia C Bauer ◽  
Isabelle B Pickles ◽  
Sara Hosseini-Farahabadi ◽  
Aruna Balgi ◽  
...  
Keyword(s):  
Premature Termination ◽  
Genetic Disorders ◽  
Premature Termination Codon ◽  
Cation Channels ◽  
Trpc Channels ◽  
Nonsense Mutations ◽  
Variable Effect ◽  
Premature Termination Codons ◽  
Ic50 Values ◽  
Nonsense Mediated Mrna Decay

Nonsense mutations, which occur in ~11% of patients with genetic disorders, introduce premature termination codons (PTCs) that lead to truncated proteins and promote nonsense-mediated mRNA decay. Aminoglycosides such as gentamicin and G418 permit PTC readthrough and so may address this problem. However, their effects are variable between patients, making clinical use of aminoglycosides challenging. In this study, we addressed the hypothesis that TRP non-selective cation channels contribute to the variable effect of aminoglycosides by controlling their cellular uptake. To attempt to identify the channel type involved, we tested AC1903, a 2-aminobenzimidazole derivative recently reported to selectively inhibit TRPC5 cation channels. AC1903 consistently suppressed G418 uptake and G418-induced PTC readthrough in the DMS-114 cell line and patient-derived JEB01 keratinocytes. In an effort to validate the suggested role of TRPC5, we tested an independent and more potent inhibitor called Pico145, which affects channels containing TRPC1, TRPC4 and TRPC5 but not other TRPCs or other channels. Unexpectedly, Pico145 was completely without effect, suggesting that AC1903 may work through other or additional targets. Consistent with this suggestion, AC1903 inhibited multiple TRPC channels including homomeric TRPC3, TRPC4, TRPC5, TRPC6 as well as concatemeric TRPC4-C1 and TRPC5-C1 channels, all with low micromolar IC50 values. It also inhibited TRPV4 channels but had weak or no effects on TRPV1 and no effect on another non-selective cation channel, PIEZO1. Overall, our study reveals a suppressor of aminoglycoside-mediated PTC readthrough (i.e., AC1903) but suggests that this compound has previously unrecognised effects. These effects require further investigation to determine the molecular mechanism by which AC1903 suppresses aminoglycoside uptake and PTC readthrough.

scholarly journals CRISPR-pass: Gene rescue of nonsense mutations using adenine base editors

2019 ◽  
Author(s):  
Choongil Lee ◽  
Dong Hyun Jo ◽  
Gue-Ho Hwang ◽  
Jihyeon Yu ◽  
Jin Hyoung Kim ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Dna Sequence ◽  
Clinical Utility ◽  
Premature Termination ◽  
Genetic Disorders ◽  
Nonsense Mutations ◽  
Premature Termination Codons ◽  
Potential Clinical Utility ◽  
Clinically Significant ◽  
Adenine Base

AbstractA nonsense mutation is a substitutive mutation in a DNA sequence that causes a premature termination during translation and produces stalled proteins resulting in dysfunction of a gene. Although it usually induces severe genetic disorders, there are no definite methods for inducing read-through of premature termination codons (PTCs). Here, we present a targeted tool for bypassing PTCs, named CRISPR-pass that uses CRISPR-mediated adenine base editors. CRISPR-pass, which should be applicable to 95.5% of clinically significant nonsense mutations in the ClinVar database, rescues protein synthesis in patient-derived fibroblasts, suggesting potential clinical utility.

scholarly journals Premature termination codon readthrough in Drosophila varies in a developmental and tissue-specific manner

2019 ◽  
Author(s):  
Yanan Chen ◽  
Tianhui Sun ◽  
Zhuo Bi ◽  
Jian-Quan Ni ◽  
Jose C. Pastor-Pareja ◽  
...  
Keyword(s):  
Life Cycle ◽  
Premature Termination ◽  
Stop Codon ◽  
Premature Termination Codon ◽  
Anatomical Site ◽  
Stop Codons ◽  
Premature Termination Codons ◽  
Nonsense Mediated Mrna Decay ◽  
Rescue Mechanism ◽  
Stop Codon Readthrough

AbstractDespite their essential function in terminating translation, readthrough of stop codons occurs more frequently than previously supposed. However, little is known about the regulation of stop codon readthrough by anatomical site and over the life cycle of animals. Here, we developed a set of reporters to measure readthrough in Drosophila melanogaster. A focused RNAi screen in whole animals identified upf1 as a mediator of readthrough, suggesting that the stop codons in the reporters were recognized as premature termination codons (PTCs). We found readthrough rates of PTCs varied significantly throughout the life cycle of flies, being highest in older adult flies. Furthermore, readthrough rates varied dramatically by tissue and, intriguingly, were highest in fly brains, specifically neurons and not glia. This was not due to differences in reporter abundance or nonsense-mediated mRNA decay (NMD) surveillance between these tissues. Overall, our data reveal temporal and spatial variation of PTC-mediated readthrough in animals, and suggest that readthrough may be a potential rescue mechanism for PTC-harboring transcripts when the NMD surveillance pathway is inhibited.

scholarly journals LQT2 nonsense mutations generate trafficking defective NH2-terminally truncated channels by the reinitiation of translation

2013 ◽  
Vol 305 (9) ◽  
pp. H1397-H1404 ◽  
Author(s):  
Matthew R. Stump ◽  
Qiuming Gong ◽  
Zhengfeng Zhou
Keyword(s):  
Ventricular Arrhythmias ◽  
Premature Termination Codon ◽  
Wild Type ◽  
Autosomal Dominant Disorder ◽  
Nonsense Mutations ◽  
Accelerated Degradation ◽  
Herg Current ◽  
Nonsense Mediated Mrna Decay ◽  
Herg Channels

The human ether-a-go-go-related gene ( hERG) encodes a voltage-activated K+ channel that contributes to the repolarization of the cardiac action potential. Long QT syndrome type 2 (LQT2) is an autosomal dominant disorder caused by mutations in hERG, and patients with LQT2 are susceptible to severe ventricular arrhythmias. We have previously shown that nonsense and frameshift LQT2 mutations caused a decrease in mutant mRNA by the nonsense-mediated mRNA decay (NMD) pathway. The Q81X nonsense mutation was recently found to be resistant to NMD. Translation of Q81X is reinitiated at Met124, resulting in the generation of NH2-terminally truncated hERG channels with altered gating properties. In the present study, we identified two additional NMD-resistant LQT2 nonsense mutations, C39X and C44X, in which translation is reinitiated at Met60. Deletion of the first 59 residues of the channel truncated nearly one-third of the highly structured Per-Arnt-Sim domain and resulted in the generation of trafficking-defective proteins and a complete loss of hERG current. Partial deletion of the Per-Arnt-Sim domain also resulted in the accelerated degradation of the mutant channel proteins. The coexpression of mutant and wild-type channels did not significantly disrupt the function and trafficking properties of wild-type hERG. Our present findings indicate that translation reinitiation may generate trafficking-defective as well as dysfunctional channels in patients with LQT2 premature termination codon mutations that occur early in the coding sequence.

scholarly journals Screening Readthrough Compounds to Suppress Nonsense Mutations: Possible Application to β-Thalassemia

2020 ◽  
Vol 9 (2) ◽  
pp. 289 ◽  
Author(s):  
Monica Borgatti ◽  
Emiliano Altamura ◽  
Francesca Salvatori ◽  
Elisabetta D’Aversa ◽  
Nicola Altamura
Keyword(s):  
Genetic Disease ◽  
Chelation Therapy ◽  
Translation Termination ◽  
Premature Termination Codon ◽  
Nonsense Suppression ◽  
Current Status ◽  
Nonsense Mutations ◽  
Nonsense Mediated Mrna Decay ◽  
Mrna Destabilization ◽  
Premature Translation Termination

Several types of thalassemia (including β039-thalassemia) are caused by nonsense mutations in genes controlling globin production, leading to premature translation termination and mRNA destabilization mediated by the nonsense mediated mRNA decay. Drugs (for instance, aminoglycosides) can be designed to suppress premature translation termination by inducing readthrough (or nonsense suppression) at the premature termination codon. These findings have introduced new hopes for the development of a pharmacologic approach to cure this genetic disease. In the present review, we first summarize the principle and current status of the chemical relief for the expression of functional proteins from genes otherwise unfruitful for the presence of nonsense mutations. Second, we compare data available on readthrough molecules for β0-thalassemia. The examples reported in the review strongly suggest that ribosomal readthrough should be considered as a therapeutic approach for the treatment of β0-thalassemia caused by nonsense mutations. Concluding, the discovery of molecules, exhibiting the property of inducing β-globin, such as readthrough compounds, is of great interest and represents a hope for several patients, whose survival will depend on the possible use of drugs rendering blood transfusion and chelation therapy unnecessary.

scholarly journals Nonsense-Mediated mRNA Decay Mutation in Aspergillus nidulans

2006 ◽  
Vol 5 (11) ◽  
pp. 1838-1846 ◽  
Author(s):  
Igor Y. Morozov ◽  
Susana Negrete-Urtasun ◽  
Joan Tilburn ◽  
Christine A. Jansen ◽  
Mark X. Caddick ◽  
...  
Keyword(s):  
Aspergillus Nidulans ◽  
Mrna Decay ◽  
Premature Termination ◽  
Regulation Of Gene Expression ◽  
Premature Termination Codon ◽  
Termination Codon ◽  
Urate Oxidase ◽  
Genes Encoding ◽  
Nonsense Mediated Mrna Decay ◽  
Nonsense Codons

ABSTRACT An Aspergillus nidulans mutation, designated nmdA1, has been selected as a partial suppressor of a frameshift mutation and shown to truncate the homologue of the Saccharomyces cerevisiae nonsense-mediated mRNA decay (NMD) surveillance component Nmd2p/Upf2p. nmdA1 elevates steady-state levels of premature termination codon-containing transcripts, as demonstrated using mutations in genes encoding xanthine dehydrogenase (hxA), urate oxidase (uaZ), the transcription factor mediating regulation of gene expression by ambient pH (pacC), and a protease involved in pH signal transduction (palB). nmdA1 can also stabilize pre-mRNA (unspliced) and wild-type transcripts of certain genes. Certain premature termination codon-containing transcripts which escape NMD are relatively stable, a feature more in common with certain nonsense codon-containing mammalian transcripts than with those in S. cerevisiae. As in S. cerevisiae, 5′ nonsense codons are more effective at triggering NMD than 3′ nonsense codons. Unlike the mammalian situation but in common with S. cerevisiae and other lower eukaryotes, A. nidulans is apparently impervious to the position of premature termination codons with respect to the 3′ exon-exon junction.

scholarly journals Nonsense Mutations in the lsa-Like Gene in Enterococcus faecalis Isolates Susceptible to Lincosamides and Streptogramins A

2003 ◽  
Vol 47 (7) ◽  
pp. 2307-2309 ◽  
Author(s):  
Julia Dina ◽  
Brigitte Malbruny ◽  
Roland Leclercq
Keyword(s):  
Enterococcus Faecalis ◽  
Premature Termination ◽  
Clinical Isolates ◽  
Intrinsic Resistance ◽  
Nonsense Mutations ◽  
Active Efflux ◽  
Premature Termination Codons

ABSTRACT The lsa gene confers intrinsic resistance to lincosamides and streptogramins A in Enterococcus faecalis, probably by active efflux. The lsa-like genes of two clinical isolates of E. faecalis susceptible to lincosamides and dalfopristin contained mutations that produced premature termination codons. Revertant mutants were obtained by selection on agar plates containing clindamycin.

scholarly journals Aminoglycoside-induced premature termination codon readthrough of COL4A5 nonsense mutations that cause Alport syndrome

2021 ◽  
Author(s):  
Kohei Omachi ◽  
Hirofumi Kai ◽  
Michel Roberge ◽  
Jeffrey H Miner
Keyword(s):  
Alport Syndrome ◽  
Premature Termination ◽  
Genetic Diseases ◽  
Premature Termination Codon ◽  
Full Length ◽  
Termination Codon ◽  
Reporter System ◽  
Nonsense Mutations ◽  
Type Iv ◽  
Genes Encoding

Alport syndrome (AS) is characterized by glomerular basement membrane (GBM) abnormalities leading to progressive glomerulosclerosis. Mutations in the COL4A3, COL4A4 or COL4A5 genes encoding type IV collagen α3α4α5 cause AS. Truncated α3, α4, and α5 chains lacking an intact COOH-terminal noncollagenous domain due to a premature termination codon (PTC) cannot assemble into heterotrimers or incorporate into the GBM. Therefore, achieving full-length protein expression is a potential therapy for AS caused by truncating nonsense mutations. Small molecule-based PTC readthrough (PTC-RT) therapy has been well studied in other genetic diseases, but whether PTC-RT is applicable to AS is unexplored. To investigate the feasibility of PTC-RT therapy in AS, we made a cDNA to express COL4A5 fused to a C-terminal NanoLuc luciferase (NLuc) to monitor full-length translation. Full-length COL4A5-NLuc produces luminescence, but mutants truncated due to a PTC do not. To screen for COL4A5 nonsense mutants susceptible to PTC-RT, we introduced 49 individual nonsense mutations found in AS patients into the COL4A5-NLuc cDNA. Luciferase assays revealed that 11 mutations (C29X, S36X, E130X, C1521X, R1563X, C1567X, W1594X, S1632X, R1683X, C1684X and K1689X) were susceptible to PTC-RT induced by G418, which is known to have high readthrough activity. Moreover, we found that some next-generation "designer" PTC-RT drugs induced RT, and RT enhancer compounds increased the efficacy of PTC-RT in a G418-susceptible PTC mutant. These results suggest that PTC-RT therapy is a feasible approach for some patients with AS. Our luciferase-based COL4A5 translation reporter system will contribute to further development of PTC-RT therapies in a personalized medicine approach to treating AS.

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