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Benslimane, Nesrine, Camille Loret, Pauline Chazelas, Frédéric Favreau, Pierre-Antoine Faye, Fabrice Lejeune, and Anne-Sophie Lia. "Readthrough Activators and Nonsense-Mediated mRNA Decay Inhibitor Molecules: Real Potential in Many Genetic Diseases Harboring Premature Termination Codons." Pharmaceuticals 17, no. 3 (February 28, 2024): 314. http://dx.doi.org/10.3390/ph17030314.
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Nonsense mutations that generate a premature termination codon (PTC) can induce both the accelerated degradation of mutated mRNA compared with the wild type version of the mRNA or the production of a truncated protein. One of the considered therapeutic strategies to bypass PTCs is their “readthrough” based on small-molecule drugs. These molecules promote the incorporation of a near-cognate tRNA at the PTC position through the native polypeptide chain. In this review, we detailed the various existing strategies organized according to pharmacological molecule types through their different mechanisms. The positive results that followed readthrough molecule testing in multiple neuromuscular disorder models indicate the potential of this approach in peripheral neuropathies.
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Baradaran-Heravi, Alireza, Aruna D. Balgi, Sara Hosseini-Farahabadi, Kunho Choi, Cristina Has, and Michel Roberge. "Effect of small molecule eRF3 degraders on premature termination codon readthrough." Nucleic Acids Research 49, no. 7 (March 25, 2021): 3692–708. http://dx.doi.org/10.1093/nar/gkab194.
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Abstract Premature termination codon (PTC) readthrough is considered a potential treatment for genetic diseases caused by nonsense mutations. High concentrations of aminoglycosides induce low levels of PTC readthrough but also elicit severe toxicity. Identifying compounds that enhance PTC readthrough by aminoglycosides or reduce their toxicity is a continuing challenge. In humans, a binary complex of eukaryotic release factors 1 (eRF1) and 3 (eRF3a or eRF3b) mediates translation termination. They also participate in the SURF (SMG1-UPF1-eRF1-eRF3) complex assembly involved in nonsense-mediated mRNA decay (NMD). We show that PTC readthrough by aminoglycoside G418 is considerably enhanced by eRF3a and eRF3b siRNAs and cereblon E3 ligase modulators CC-885 and CC-90009, which induce proteasomal degradation of eRF3a and eRF3b. eRF3 degradation also reduces eRF1 levels and upregulates UPF1 and selectively stabilizes TP53 transcripts bearing a nonsense mutation over WT, indicating NMD suppression. CC-90009 is considerably less toxic than CC-885 and it enhances PTC readthrough in combination with aminoglycosides in mucopolysaccharidosis type I-Hurler, late infantile neuronal ceroid lipofuscinosis, Duchenne muscular dystrophy and junctional epidermolysis bullosa patient-derived cells with nonsense mutations in the IDUA, TPP1, DMD and COL17A1 genes, respectively. Combination of CC-90009 with aminoglycosides such as gentamicin or ELX-02 may have potential for PTC readthrough therapy.
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Perriera, Riccardo, Emanuele Vitale, Ivana Pibiri, Pietro Salvatore Carollo, Davide Ricci, Federica Corrao, Ignazio Fiduccia, et al. "Readthrough Approach Using NV Translational Readthrough-Inducing Drugs (TRIDs): A Study of the Possible Off-Target Effects on Natural Termination Codons (NTCs) on TP53 and Housekeeping Gene Expression." International Journal of Molecular Sciences 24, no. 20 (October 11, 2023): 15084. http://dx.doi.org/10.3390/ijms242015084.
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Nonsense mutations cause several genetic diseases such as cystic fibrosis, Duchenne muscular dystrophy, β-thalassemia, and Shwachman–Diamond syndrome. These mutations induce the formation of a premature termination codon (PTC) inside the mRNA sequence, resulting in the synthesis of truncated polypeptides. Nonsense suppression therapy mediated by translational readthrough-inducing drugs (TRIDs) is a promising approach to correct these genetic defects. TRIDs generate a ribosome miscoding of the PTC named “translational readthrough” and restore the synthesis of full-length and potentially functional proteins. The new oxadiazole-core TRIDs NV848, NV914, and NV930 (NV) showed translational readthrough activity in nonsense-related in vitro systems. In this work, the possible off-target effect of NV molecules on natural termination codons (NTCs) was investigated. Two different in vitro approaches were used to assess if the NV molecule treatment induces NTC readthrough: (1) a study of the translational-induced p53 molecular weight and functionality; (2) the evaluation of two housekeeping proteins’ (Cys-C and β2M) molecular weights. Our results showed that the treatment with NV848, NV914, or NV930 did not induce any translation alterations in both experimental systems. The data suggested that NV molecules have a specific action for the PTCs and an undetectable effect on the NTCs.
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Hosseini-Farahabadi, Sara, Alireza Baradaran-Heravi, Carla Zimmerman, Kunho Choi, Stephane Flibotte, and Michel Roberge. "Small molecule Y-320 stimulates ribosome biogenesis, protein synthesis, and aminoglycoside-induced premature termination codon readthrough." PLOS Biology 19, no. 5 (May 3, 2021): e3001221. http://dx.doi.org/10.1371/journal.pbio.3001221.
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Premature termination codons (PTC) cause over 10% of genetic disease cases. Some aminoglycosides that bind to the ribosome decoding center can induce PTC readthrough and restore low levels of full-length functional proteins. However, concomitant inhibition of protein synthesis limits the extent of PTC readthrough that can be achieved by aminoglycosides like G418. Using a cell-based screen, we identified a small molecule, the phenylpyrazoleanilide Y-320, that potently enhances TP53, DMD, and COL17A1 PTC readthrough by G418. Unexpectedly, Y-320 increased cellular protein levels and protein synthesis, measured by SYPRO Ruby protein staining and puromycin labeling, as well as ribosome biogenesis measured using antibodies to rRNA and ribosomal protein S6. Y-320 did not increase the rate of translation elongation and it exerted its effects independently of mTOR signaling. At the single cell level, exposure to Y-320 and G418 increased ribosome content and protein synthesis which correlated strongly with PTC readthrough. As a single agent, Y-320 did not affect translation fidelity measured using a luciferase reporter gene but it enhanced misincorporation by G418. RNA-seq data showed that Y-320 up-regulated the expression of CXC chemokines CXCL10, CXCL8, CXCL2, CXCL11, CXCL3, CXCL1, and CXCL16. Several of these chemokines exert their cellular effects through the receptor CXCR2 and the CXCR2 antagonist SB225002 reduced cellular protein levels and PTC readthrough in cells exposed to Y-320 and G418. These data show that the self-limiting nature of PTC readthrough by G418 can be compensated by Y-320, a potent enhancer of PTC readthrough that increases ribosome biogenesis and protein synthesis. They also support a model whereby increased PTC readthrough is enabled by increased protein synthesis mediated by an autocrine chemokine signaling pathway. The findings also raise the possibility that inflammatory processes affect cellular propensity to readthrough agents and that immunomodulatory drugs like Y-320 might find application in PTC readthrough therapy.
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Simmons, Zoe R., Amanda Sherwood, Selena Li, Sylvie Garneau-Tsodikova, and Matthew Gentry. "2348 Lafora disease premature termination codons (PTCs) are likely candidates for suppression by aminoglycosides." Journal of Clinical and Translational Science 2, S1 (June 2018): 16–17. http://dx.doi.org/10.1017/cts.2018.90.
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OBJECTIVES/SPECIFIC AIMS: A small molecule therapy is within reach to treat a molecular mechanism known to result in thousands of fatal diseases. For 10% of patients with a genetic disease, a nonsense/STOP mutation/premature termination codon (PTC) is the underlying cause of their malady. PTCs prematurely stop protein synthesis and yield truncated proteins. Truncated proteins typically provide little to no proper function or activity and are rapidly degraded; thus, disease is imminent. Recent work has demonstrated that small molecules including aminoglycosides can cause the ribosome to readthrough these PTCs. Thus, PTC readthrough with small molecules is a very attractive approach for treating diseases caused by PTCs. Small molecules that promote readthrough act on the ribosome and induce a ribosomal conformational change. In this conformation, the PTC is not recognized by the translational machinery and an amino acid is incorporated into the growing peptide chain, thus protein synthesis continues and does not stop. The use of a single small molecule to readthrough various PTC mutations has been repeatedly effective for in vitro studies and some of these have progressed to clinical trials. Although there has been success in defining these small molecules, the field has discovered that every PTC is unique and likely requires a different small molecule. Thus, developing a cell culture model to test read-through of Lafora PTCs and the functionality of the protein product is the first step to developing a readthrough therapy for a LD. METHODS/STUDY POPULATION: Method for in vitro quantification of readthrough: 24 hours before transfection, HEK293 cells were split in 6-well plates. On the following day, approximately 60% confluence, the cells were transiently transfected with the WT or PTC mutated constructs using Polyethylenimine HCl MAX. Cells were transfected with a total amount of 0.35 μg DNA/well and 2 μl Polyethylenimine HCl MAX/well. Four hours later, the transfection medium was removed and replaced with fresh medium, without streptomycin and penicillin. The fresh media contained gentamicin diluted to the indicated concentration per well. Fresh gentamicin-containing medium was replaced after 24 hours. After 48 hours, lysates were collected in 100 μL mRIPA supplemented with protease inhibitors for each construct. The lysates were run on a western blot and the N-terminal was probed with anti-FLAG. A malachite green phosphatase assay to measure inorganic phosphate release from phospho-glucans, that is glycogen or LBs. Glycogen is used in this laforin bioassay as the biologically relevant substrate in order to determine the specific activity of the readthrough products. All reactions are incubated for 40 minute the absorbance is measured at 620 nm and the pmoles of phosphate released/min/nmol protein was calculated using a standard curve. RESULTS/ANTICIPATED RESULTS: HEK293 cells were transfected with MeCP2 R241X, laforin R241X, or laforin WT NT-FLAG construct, treated with different concentrations of gentamicin for 48 hours, and laforin levels were assessed by Western analysis with anti-FLAG. HEK293 cells were transfected with WT laforin or a laforin PTC CT-FLAG construct, treated with different concentrations of gentamicin for 48 hours, and laforin levels were assessed by Western analysis with anti-FLAG. B. Quantification of read-through for PTC experiments. *p-value≤0.001. #p-value≤0.001. Schematic of laforin bioassay. The assay has been performed with human and mouse tissue as well as cultured cells. B. Laforin bioassay results using laforin from PTC experiment. **p-value≤0.001. *p-value≤0.01. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that gentamicin is not only responsible for inducing readthrough of the PTC mutations, but also for promoting translation of fully functional laforin. Therefore, our in vitro system for the analysis of PTC readthrough of laforin will be useful for determining which PTC mutations are suppressible with gentamicin or other small molecules, in what quantities laforin is recovered from PTC mutations, and if the protein products possess the appropriate enzymatic function.
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Pranke, Iwona, Laure Bidou, Natacha Martin, Sandra Blanchet, Aurélie Hatton, Sabrina Karri, David Cornu, et al. "Factors influencing readthrough therapy for frequent cystic fibrosis premature termination codons." ERJ Open Research 4, no. 1 (January 2018): 00080–2017. http://dx.doi.org/10.1183/23120541.00080-2017.
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Premature termination codons (PTCs) are generally associated with severe forms of genetic diseases. Readthrough of in-frame PTCs using small molecules is a promising therapeutic approach. Nonetheless, the outcome of preclinical studies has been low and variable. Treatment efficacy depends on: 1) the level of drug-induced readthrough, 2) the amount of target transcripts, and 3) the activity of the recoded protein. The aim of the present study was to identify, in the cystic fibrosis transmembrane conductance regulator (CFTR) model, recoded channels from readthrough therapy that may be enhanced using CFTR modulators.First, drug-induced readthrough of 15 PTCs was measured using a dual reporter system under basal conditions and in response to gentamicin and negamycin. Secondly, exon skipping associated with these PTCs was evaluated with a minigene system. Finally, incorporated amino acids were identified by mass spectrometry and the function of the predicted recoded CFTR channels corresponding to these 15 PTCs was measured. Nonfunctional channels were subjected to CFTR-directed ivacaftor-lumacaftor treatments.The results demonstrated that CFTR modulators increased activity of recoded channels, which could also be confirmed in cells derived from a patient.In conclusion, this work will provide a framework to adapt treatments to the patient's genotype by identifying the most efficient molecule for each PTC and the recoded channels needing co-therapies to rescue channel function.
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Mathews, Paul. "32329 A novel mouse model of Ataxia Telangiectasia for testing small molecule readthrough compounds." Journal of Clinical and Translational Science 5, s1 (March 2021): 11. http://dx.doi.org/10.1017/cts.2021.430.
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ABSTRACT IMPACT: Small molecule readthrough compounds are a promising therapeutic with the potential to overcome nonsense mutations thereby enabling the production of functional ATM protein in patients with Ataxia Telangiectasia OBJECTIVES/GOALS: To generate a novel mouse model of Ataxia-Telangiectasia for testing small molecule readthrough compounds that both expresses a clinically relevant nonsense mutation and recapitulates the major symptoms of the disease, including a progressive loss of motor coordination not previously observed in prior A-T animal models. METHODS/STUDY POPULATION: Using a double-hit strategy to increase genotoxic stress, we generated a novel A-T mouse model that expresses a clinically relevant (c.103C>T) mutation in the Atm gene and a knockout of the functionally related Aptx gene. We then characterized the mouse across multiple domains related to the various symptoms related to the disease. This includes examination of survivability, immunologic function, cancer prevalence, and motor behavior and its associated cerebellar dysfunction and atrophy. Lastly, we tested the ability of small molecule readthrough compounds to enable production of ATM from tissue explants extracted from these ATM deficient mice. RESULTS/ANTICIPATED RESULTS: The double mutant mice display reduced survivability compared to control mice (53% vs. 97%; p<0.0001), dying at a clinically relevant rate of about 30% from thymomas. At postnatal day 400 (P400), only AtmR35X/R35X; Aptx-/- mice, and none of the controls expressing at least one wildtype Atm or Aptx gene develop a motor behavioral deficits that are associate with reduced Purkinje neuron diameter (8.0 ±0.4 µm vs. 9.92 ±0.5; p<0.01) and density (4.3 ±0.2 vs. 6.0 ±0.3 per 100 µm; p<0.05) as well as cerebellar atrophy (cerebellum/forebrain area 0.26 ±0.01 vs. 0.31 ±0.01; p<0.001). ATM deficient mice also display disrupted thymocyte development and metabolic function. When exposed to small molecular readthrough compounds, greater than 50% of the ATM protein is restored. DISCUSSION/SIGNIFICANCE OF FINDINGS: We have created a novel, clinically relevant A-T mouse model that develops a severe ataxia associated with changes in cerebellar function and atrophy as well as demonstrate the potential of SMRT compounds as an A-T therapeutic.
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Kuang, Lisha, Kei Hashimoto, Eric J. Huang, Matthew S. Gentry, and Haining Zhu. "Frontotemporal dementia non-sense mutation of progranulin rescued by aminoglycosides." Human Molecular Genetics 29, no. 4 (January 8, 2020): 624–34. http://dx.doi.org/10.1093/hmg/ddz280.
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Abstract Frontotemporal dementia (FTD) is an early onset dementia characterized by progressive atrophy of the frontal and/or temporal lobes. FTD is highly heritable with mutations in progranulin accounting for 5–26% of cases in different populations. Progranulin is involved in endocytosis, secretion and lysosomal processes, but its functions under physiological and pathological conditions remains to be defined. Many FTD-causing non-sense progranulin mutations contain a premature termination codon (PTC), thus progranulin haploinsufficiency has been proposed as a major disease mechanism. Currently, there is no effective FTD treatment or therapy. Aminoglycosides are a class of antibiotics that possess a less-known function to induce eukaryotic ribosomal readthrough of PTCs to produce a full-length protein. The aminoglycoside-induced readthrough strategy has been utilized to treat multiple human diseases caused by PTCs. In this study, we tested the only clinically approved readthrough small molecule PTC124 and 11 aminoglycosides in a cell culture system on four PTCs responsible for FTD or a related neurodegenerative disease amyotrophic lateral sclerosis. We found that the aminoglycosides G418 and gentamicin rescued the expression of the progranulin R493X mutation. G418 was more effective than gentamicin (~50% rescue versus <10%), and the effect was dose- and time-dependent. The progranulin readthrough protein displayed similar subcellular localization as the wild-type progranulin protein. These data provide an exciting proof-of-concept that aminoglycosides or other readthrough-promoting compounds are a therapeutic avenue for familial FTD caused by progranulin PTC mutations.
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Wagner, Roland N., Michael Wießner, Andreas Friedrich, Johanna Zandanell, Hannelore Breitenbach-Koller, and Johann W. Bauer. "Emerging Personalized Opportunities for Enhancing Translational Readthrough in Rare Genetic Diseases and Beyond." International Journal of Molecular Sciences 24, no. 7 (March 23, 2023): 6101. http://dx.doi.org/10.3390/ijms24076101.
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Nonsense mutations trigger premature translation termination and often give rise to prevalent and rare genetic diseases. Consequently, the pharmacological suppression of an unscheduled stop codon represents an attractive treatment option and is of high clinical relevance. At the molecular level, the ability of the ribosome to continue translation past a stop codon is designated stop codon readthrough (SCR). SCR of disease-causing premature termination codons (PTCs) is minimal but small molecule interventions, such as treatment with aminoglycoside antibiotics, can enhance its frequency. In this review, we summarize the current understanding of translation termination (both at PTCs and at cognate stop codons) and highlight recently discovered pathways that influence its fidelity. We describe the mechanisms involved in the recognition and readthrough of PTCs and report on SCR-inducing compounds currently explored in preclinical research and clinical trials. We conclude by reviewing the ongoing attempts of personalized nonsense suppression therapy in different disease contexts, including the genetic skin condition epidermolysis bullosa.
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Liu, Yi-Lin, Paris Margaritis, Fayaz Khazi, Harre Downey, Stephan Kadauke, Nicole Hasbrouck, Josephine Sheedy, Ellen Welch, Marla Weetall, and Katherine A. High. "Nonsense Suppression Approaches in Treating Hemophilia." Blood 112, no. 11 (November 16, 2008): 512. http://dx.doi.org/10.1182/blood.v112.11.512.512.
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Abstract Genetic diseases can result from nonsense mutations that cause premature translation termination. Nonsense mutations account for ~10–15% of all cases of hemophilia A and B and this population may benefit from small molecule-induced readthrough of nonsense codons. Recent studies document the ability of an orally bioavailable small molecule, PTC124, to facilitate dose-dependent readthrough of nonsense codons in a variety of in vitro and in vivo systems, including reporter gene constructs, mdx mice (a murine model of Duchenne muscular dystrophy; Nature447:87–91, 2007), and in a mouse model of cystic fibrosis (PNAS105: 2064–2069, 2008). PTC124 was well-tolerated in healthy human volunteers (J. Clin. Pharmacol.47:430–444, 2007) and in patients with cystic fibrosis (The Lancet, online August 21, 2008). We previously constructed murine models of severe hemophilia B with nonsense mutations in a transgenic human factor IX (hF.IX) gene resulting in nonsense codons at amino acid positions 29 or 338 (R29X and R338X; Blood104:2767–2774, 2004). These mice have no circulating hF.IX detectable by ELISA. To evaluate the effect of small molecules on readthrough in the hemophilia B model, we administered PTC124 to mice carrying the R338X mutation by subcutaneous injection for three days. Measurable plasma levels of hF.IX were detected by ELISA (n=3) and high levels of hF.IX were detected in the liver by immunohistochemistry in the single mouse that was sacrificed. Following oral administration of PTC124 to R338X mice for three days, 20% of mice demonstrated detectable circulating hF.IX levels in the range of 3–5 ng/mL. In a separate experiment, we administered another small molecule, PTC-EMK, by IP injection for three days to R338X mice. With this regimen, 40% of mice showed detectable circulating hF.IX levels. To examine the effect of PTC124 or PTC-EMK on other nonsense mutations, we prepared ten of the most frequently reported nonsense mutations in the hF.IX gene (R29X, R116X, W194X, R248X, R252X, Y266X, W310X, R333X, R338X, and W407X) and transiently transfected these constructs into HEK293 cells. The cells were treated with PTC124 or PTCEMK at various concentrations for 72 hrs and the level of hF.IX protein in the medium was quantified using the ELISA. Of the nonsense containing constructs analyzed, 6/10 mutants showed positive responses with the largest response noted for cells containing the R29X mutation treated with PTC-EMK. In summary, our results demonstrate the ability of PTC124 and PTC-EMK to read through nonsense codons in the hF.IX mRNA and support the potential use of orally bioavailable small molecules as a therapeutic option for patients with hemophilia due to nonsense mutations.
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Peh, J., T. Miyauchi, M. Takeda, S. Suzuki, H. Ujiie, and T. Nomura. "172 Discovery of small molecule compounds with readthrough potency at premature termination codon." Journal of Investigative Dermatology 141, no. 10 (October 2021): S177. http://dx.doi.org/10.1016/j.jid.2021.08.176.
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Bhattacharya, Arpan, Mikel D. Ghelfi, Xiaonan Cui, Clark Fritsch, Hong Li, Barry S. Cooperman, and Yale E. Goldman. "Translational readthrough-inducing drug (TRID) effects on eukaryotic termination investigated at the single-molecule level." Biophysical Journal 122, no. 3 (February 2023): 490a. http://dx.doi.org/10.1016/j.bpj.2022.11.2618.
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Roy, Bijoyita, Westley J. Friesen, Yuki Tomizawa, John D. Leszyk, Jin Zhuo, Briana Johnson, Jumana Dakka, et al. "Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression." Proceedings of the National Academy of Sciences 113, no. 44 (October 4, 2016): 12508–13. http://dx.doi.org/10.1073/pnas.1605336113.
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A premature termination codon (PTC) in the ORF of an mRNA generally leads to production of a truncated polypeptide, accelerated degradation of the mRNA, and depression of overall mRNA expression. Accordingly, nonsense mutations cause some of the most severe forms of inherited disorders. The small-molecule drug ataluren promotes therapeutic nonsense suppression and has been thought to mediate the insertion of near-cognate tRNAs at PTCs. However, direct evidence for this activity has been lacking. Here, we expressed multiple nonsense mutation reporters in human cells and yeast and identified the amino acids inserted when a PTC occupies the ribosomal A site in control, ataluren-treated, and aminoglycoside-treated cells. We find that ataluren’s likely target is the ribosome and that it produces full-length protein by promoting insertion of near-cognate tRNAs at the site of the nonsense codon without apparent effects on transcription, mRNA processing, mRNA stability, or protein stability. The resulting readthrough proteins retain function and contain amino acid replacements similar to those derived from endogenous readthrough, namely Gln, Lys, or Tyr at UAA or UAG PTCs and Trp, Arg, or Cys at UGA PTCs. These insertion biases arise primarily from mRNA:tRNA mispairing at codon positions 1 and 3 and reflect, in part, the preferred use of certain nonstandard base pairs, e.g., U-G. Ataluren’s retention of similar specificity of near-cognate tRNA insertion as occurs endogenously has important implications for its general use in therapeutic nonsense suppression.
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Friesen, Westley J., Briana Johnson, Jairo Sierra, Jin Zhuo, Priya Vazirani, Xiaojiao Xue, Yuki Tomizawa, et al. "The minor gentamicin complex component, X2, is a potent premature stop codon readthrough molecule with therapeutic potential." PLOS ONE 13, no. 10 (October 25, 2018): e0206158. http://dx.doi.org/10.1371/journal.pone.0206158.
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Dmitriev, S. E., D. O. Vladimirov, and K. A. Lashkevich. "A Quick Guide to Small-Molecule Inhibitors of Eukaryotic Protein Synthesis." Biochemistry (Moscow) 85, no. 11 (November 2020): 1389–421. http://dx.doi.org/10.1134/s0006297920110097.
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Abstract Eukaryotic ribosome and cap-dependent translation are attractive targets in the antitumor, antiviral, anti-inflammatory, and antiparasitic therapies. Currently, a broad array of small-molecule drugs is known that specifically inhibit protein synthesis in eukaryotic cells. Many of them are well-studied ribosome-targeting antibiotics that block translocation, the peptidyl transferase center or the polypeptide exit tunnel, modulate the binding of translation machinery components to the ribosome, and induce miscoding, premature termination or stop codon readthrough. Such inhibitors are widely used as anticancer, anthelmintic and antifungal agents in medicine, as well as fungicides in agriculture. Chemicals that affect the accuracy of stop codon recognition are promising drugs for the nonsense suppression therapy of hereditary diseases and restoration of tumor suppressor function in cancer cells. Other compounds inhibit aminoacyl-tRNA synthetases, translation factors, and components of translation-associated signaling pathways, including mTOR kinase. Some of them have antidepressant, immunosuppressive and geroprotective properties. Translation inhibitors are also used in research for gene expression analysis by ribosome profiling, as well as in cell culture techniques. In this article, we review well-studied and less known inhibitors of eukaryotic protein synthesis (with the exception of mitochondrial and plastid translation) classified by their targets and briefly describe the action mechanisms of these compounds. We also present a continuously updated database (http://eupsic.belozersky.msu.ru/) that currently contains information on 370 inhibitors of eukaryotic protein synthesis.
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Baradaran-Heravi, Alireza, Claudia C. Bauer, Isabelle B. Pickles, Sara Hosseini-Farahabadi, Aruna D. Balgi, Kunho Choi, Deborah M. Linley, David J. Beech, Michel Roberge, and Robin S. Bon. "Nonselective TRPC channel inhibition and suppression of aminoglycoside-induced premature termination codon readthrough by the small molecule AC1903." Journal of Biological Chemistry 298, no. 2 (February 2022): 101546. http://dx.doi.org/10.1016/j.jbc.2021.101546.
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Blanco-Luquin, Idoia, Blanca Acha, Amaya Urdánoz-Casado, Eva Gómez-Orte, Miren Roldan, Diego R. Pérez-Rodríguez, Juan Cabello, and Maite Mendioroz. "NXN Gene Epigenetic Changes in an Adult Neurogenesis Model of Alzheimer’s Disease." Cells 11, no. 7 (March 22, 2022): 1069. http://dx.doi.org/10.3390/cells11071069.
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In view of the proven link between adult hippocampal neurogenesis (AHN) and learning and memory impairment, we generated a straightforward adult neurogenesis in vitro model to recapitulate DNA methylation marks in the context of Alzheimer’s disease (AD). Neural progenitor cells (NPCs) were differentiated for 29 days and Aβ peptide 1–42 was added. mRNA expression of Neuronal Differentiation 1 (NEUROD1), Neural Cell Adhesion Molecule 1 (NCAM1), Tubulin Beta 3 Class III (TUBB3), RNA Binding Fox-1 Homolog 3 (RBFOX3), Calbindin 1 (CALB1), and Glial Fibrillary Acidic Protein (GFAP) was determined by RT-qPCR to characterize the culture and framed within the multistep process of AHN. Hippocampal DNA methylation marks previously identified in Contactin-Associated Protein 1 (CNTNAP1), SEPT5-GP1BB Readthrough (SEPT5-GP1BB), T-Box Transcription Factor 5 (TBX5), and Nucleoredoxin (NXN) genes were profiled by bisulfite pyrosequencing or bisulfite cloning sequencing; mRNA expression was also measured. NXN outlined a peak of DNA methylation overlapping type 3 neuroblasts. Aβ-treated NPCs showed transient decreases of mRNA expression for SEPT5-GP1BB and NXN on day 9 or 19 and an increase in DNA methylation on day 29 for NXN. NXN and SEPT5-GP1BB may reflect alterations detected in the brain of AD human patients, broadening our understanding of this disease.
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Gemmati, Donato, Elisabetta D’Aversa, Bianca Antonica, Miriana Grisafi, Francesca Salvatori, Stefano Pizzicotti, Patrizia Pellegatti, et al. "Gene Dosage of F5 c.3481C>T Stop-Codon (p.R1161Ter) Switches the Clinical Phenotype from Severe Thrombosis to Recurrent Haemorrhage: Novel Hypotheses for Readthrough Strategy." Genes 15, no. 4 (March 29, 2024): 432. http://dx.doi.org/10.3390/genes15040432.
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Inherited defects in the genes of blood coagulation essentially express the severity of the clinical phenotype that is directly correlated to the number of mutated alleles of the candidate leader gene (e.g., heterozygote vs. homozygote) and of possible additional coinherited traits. The F5 gene, which codes for coagulation factor V (FV), plays a two-faced role in the coagulation cascade, exhibiting both procoagulant and anticoagulant functions. Thus, defects in this gene can be predisposed to either bleeding or thrombosis. A Sanger sequence analysis detected a premature stop-codon in exon 13 of the F5 gene (c.3481C>T; p.R1161Ter) in several members of a family characterised by low circulating FV levels and contrasting clinical phenotypes. The propositus, a 29 y.o. male affected by recurrent haemorrhages, was homozygous for the F5 stop-codon and for the F5 c.1691G>A (p.R506Q; FV-Leiden) inherited from the heterozygous parents, which is suggestive of combined cis-segregation. The homozygous condition of the stop-codon completely abolished the F5 gene expression in the propositus (FV:Ag < 1%; FV:C < 1%; assessed by ELISA and PT-based one-stage clotting assay respectively), removing, in turn, any chance for FV-Leiden to act as a prothrombotic molecule. His father (57 y.o.), characterised by severe recurrent venous thromboses, underwent a complete molecular thrombophilic screening, revealing a heterozygous F2 G20210A defect, while his mother (56 y.o.), who was negative for further common coagulation defects, reported fully asymptomatic anamnesis. To dissect these conflicting phenotypes, we performed the ProC®Global (Siemens Helthineers) coagulation test aimed at assessing the global pro- and anticoagulant balance of each family member, investigating the responses to the activated protein C (APC) by means of an APC-sensitivity ratio (APC-sr). The propositus had an unexpectedly poor response to APC (APC-sr: 1.09; n.v. > 2.25), and his father and mother had an APC-sr of 1.5 and 2.0, respectively. Although ProC®Global prevalently detects the anticoagulant side of FV, the exceptionally low APC-sr of the propositus and his discordant severe–moderate haemorrhagic phenotype could suggest a residual expression of mutated FV p.506QQ through a natural readthrough or possible alternative splicing mechanisms. The coagulation pathway may be physiologically rebalanced through natural and induced strategies, and the described insights might be able to track the design of novel treatment approaches and rebalancing molecules.
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Murru, S., G. Loudianos, M. Deiana, C. Camaschella, GV Sciarratta, S. Agosti, MI Parodi, P. Cerruti, A. Cao, and M. Pirastu. "Molecular characterization of beta-thalassemia intermedia in patients of Italian descent and identification of three novel beta-thalassemia mutations." Blood 77, no. 6 (March 15, 1991): 1342–47. http://dx.doi.org/10.1182/blood.v77.6.1342.1342.
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Abstract In this study, we have defined by dot-blot analysis with allelic specific oligonucleotide probes or direct sequencing on amplified DNA the beta-thalassemia mutations in a large group of patients (23) of Italian descent with thalassemia intermedia. These patients had one parent with either the silent beta-thalassemia carrier phenotype or borderline-normal hemoglobin A2 (HbA2) levels (2.5% to 3.5%). Nearly all were genetic compounds for a severe beta-thalassemia mutation and a beta-thalassemia mutation associated with high residual output of beta- globin chains (beta + intervening sequence [IVS]-I-nt6, beta -87, beta - 101), indicating that inheritance of a mild beta-thalassemia allele, even in a single dose, is the most common molecular mechanism producing thalassemia intermedia in the Italian population. In three cases, in whom we failed to define by dot-blot analysis the mutations, we sequenced the beta + globin gene and found three novel beta-thalassemia mutations, which are certainly very rare because they have been hitherto detected solely in a single patient. These mutations consist of: (1) a T-A substitution at position 2 of IVS-I, in a patient compound heterozygote for this mutation and the -87 promoter mutation; (2) a G-C substitution at position 844 of IVS-II, in a patient heterozygous for this mutation who showed normal sequences at the in trans beta-globin gene (The reason for the presence of clinical manifestations in a beta-thalassemia heterozygote has not been defined.); and (3) a deletion of one nucleotide (-T) at codon 126, resulting in a frameshift and readthrough of the 5′ untranslated region and most likely producing an elongated Hb molecule of 156 amino acid residues, in a patient heterozygous for this mutation with normal beta- globin gene sequences at the other locus.
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Murru, S., G. Loudianos, M. Deiana, C. Camaschella, GV Sciarratta, S. Agosti, MI Parodi, P. Cerruti, A. Cao, and M. Pirastu. "Molecular characterization of beta-thalassemia intermedia in patients of Italian descent and identification of three novel beta-thalassemia mutations." Blood 77, no. 6 (March 15, 1991): 1342–47. http://dx.doi.org/10.1182/blood.v77.6.1342.bloodjournal7761342.
Full textAbstract:
In this study, we have defined by dot-blot analysis with allelic specific oligonucleotide probes or direct sequencing on amplified DNA the beta-thalassemia mutations in a large group of patients (23) of Italian descent with thalassemia intermedia. These patients had one parent with either the silent beta-thalassemia carrier phenotype or borderline-normal hemoglobin A2 (HbA2) levels (2.5% to 3.5%). Nearly all were genetic compounds for a severe beta-thalassemia mutation and a beta-thalassemia mutation associated with high residual output of beta- globin chains (beta + intervening sequence [IVS]-I-nt6, beta -87, beta - 101), indicating that inheritance of a mild beta-thalassemia allele, even in a single dose, is the most common molecular mechanism producing thalassemia intermedia in the Italian population. In three cases, in whom we failed to define by dot-blot analysis the mutations, we sequenced the beta + globin gene and found three novel beta-thalassemia mutations, which are certainly very rare because they have been hitherto detected solely in a single patient. These mutations consist of: (1) a T-A substitution at position 2 of IVS-I, in a patient compound heterozygote for this mutation and the -87 promoter mutation; (2) a G-C substitution at position 844 of IVS-II, in a patient heterozygous for this mutation who showed normal sequences at the in trans beta-globin gene (The reason for the presence of clinical manifestations in a beta-thalassemia heterozygote has not been defined.); and (3) a deletion of one nucleotide (-T) at codon 126, resulting in a frameshift and readthrough of the 5′ untranslated region and most likely producing an elongated Hb molecule of 156 amino acid residues, in a patient heterozygous for this mutation with normal beta- globin gene sequences at the other locus.
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Lombardi, Silvia, Maria Francesca Testa, Mirko Pinotti, and Alessio Branchini. "Molecular Insights into Determinants of Translational Readthrough and Implications for Nonsense Suppression Approaches." International Journal of Molecular Sciences 21, no. 24 (December 11, 2020): 9449. http://dx.doi.org/10.3390/ijms21249449.
Full textAbstract:
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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Borgatti, Monica, Emiliano Altamura, Francesca Salvatori, Elisabetta D’Aversa, and Nicola Altamura. "Screening Readthrough Compounds to Suppress Nonsense Mutations: Possible Application to β-Thalassemia." Journal of Clinical Medicine 9, no. 2 (January 21, 2020): 289. http://dx.doi.org/10.3390/jcm9020289.
Full textAbstract:
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.
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Wittenstein, Amnon, Michal Caspi, Ido Rippin, Orna Elroy-Stein, Hagit Eldar-Finkelman, Sven Thoms, and Rina Rosin-Arbesfeld. "Nonsense mutation suppression is enhanced by targeting different stages of the protein synthesis process." PLOS Biology 21, no. 11 (November 9, 2023): e3002355. http://dx.doi.org/10.1371/journal.pbio.3002355.
Full textAbstract:
The introduction of premature termination codons (PTCs), as a result of splicing defects, insertions, deletions, or point mutations (also termed nonsense mutations), lead to numerous genetic diseases, ranging from rare neuro-metabolic disorders to relatively common inheritable cancer syndromes and muscular dystrophies. Over the years, a large number of studies have demonstrated that certain antibiotics and other synthetic molecules can act as PTC suppressors by inducing readthrough of nonsense mutations, thereby restoring the expression of full-length proteins. Unfortunately, most PTC readthrough-inducing agents are toxic, have limited effects, and cannot be used for therapeutic purposes. Thus, further efforts are required to improve the clinical outcome of nonsense mutation suppressors. Here, by focusing on enhancing readthrough of pathogenic nonsense mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, we show that disturbing the protein translation initiation complex, as well as targeting other stages of the protein translation machinery, enhances both antibiotic and non-antibiotic-mediated readthrough of nonsense mutations. These findings strongly increase our understanding of the mechanisms involved in nonsense mutation readthrough and facilitate the development of novel therapeutic targets for nonsense suppression to restore protein expression from a large variety of disease-causing mutated transcripts.
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Chkuaseli, Tamari, and K. Andrew White. "Complex and simple translational readthrough signals in pea enation mosaic virus 1 and potato Leafroll virus, respectively." PLOS Pathogens 18, no. 9 (September 29, 2022): e1010888. http://dx.doi.org/10.1371/journal.ppat.1010888.
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Different essential viral proteins are translated via programmed stop codon readthrough. Pea enation mosaic virus 1 (PEMV1) and potato leafroll virus (PLRV) are related positive-sense RNA plant viruses in the family Solemoviridae, and are type members of the Enamovirus and Polerovirus genera, respectively. Both use translational readthrough to express a C-terminally extended minor capsid protein (CP), termed CP-readthrough domain (CP-RTD), from a viral subgenomic mRNA that is transcribed during infections. Limited incorporation of CP-RTD subunits into virus particles is essential for aphid transmission, however the functional readthrough structures that mediate CP-RTD translation have not yet been defined. Through RNA solution structure probing, RNA secondary structure modeling, site-directed mutagenesis, and functional in vitro and in vivo analyses, we have investigated in detail the readthrough elements and complex structure involved in expression of CP-RTD in PEMV1, and assessed and deduced a comparatively simpler readthrough structure for PLRV. Collectively, this study has (i) generated the first higher-order RNA structural models for readthrough elements in an enamovirus and a polerovirus, (ii) revealed a stark contrast in the complexity of readthrough structures in these two related viruses, (iii) provided compelling experimental evidence for the strict requirement for long-distance RNA-RNA interactions in generating the active readthrough signals, (iv) uncovered what could be considered the most complex readthrough structure reported to date, that for PEMV1, and (v) proposed plausible assembly pathways for the formation of the elaborate PEMV1 and simple PLRV readthrough structures. These findings notably advance our understanding of this essential mode of gene expression in these agriculturally important plant viruses.
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Pibiri, Ivana, Raffaella Melfi, Marco Tutone, Aldo Di Leonardo, Andrea Pace, and Laura Lentini. "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 21, no. 17 (September 3, 2020): 6420. http://dx.doi.org/10.3390/ijms21176420.
Full textAbstract:
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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Baradaran-Heravi, Alireza, Jürgen Niesser, Aruna D. Balgi, Kunho Choi, Carla Zimmerman, Andrew P. South, Hilary J. Anderson, Natalie C. Strynadka, Marcel B. Bally, and Michel Roberge. "Gentamicin B1 is a minor gentamicin component with major nonsense mutation suppression activity." Proceedings of the National Academy of Sciences 114, no. 13 (March 13, 2017): 3479–84. http://dx.doi.org/10.1073/pnas.1620982114.
Full textAbstract:
Nonsense mutations underlie about 10% of rare genetic disease cases. They introduce a premature termination codon (PTC) and prevent the formation of full-length protein. Pharmaceutical gentamicin, a mixture of several related aminoglycosides, is a frequently used antibiotic in humans that can induce PTC readthrough and suppress nonsense mutations at high concentrations. However, testing of gentamicin in clinical trials has shown that safe doses of this drug produce weak and variable readthrough activity that is insufficient for use as therapy. In this study we show that the major components of pharmaceutical gentamicin lack PTC readthrough activity but the minor component gentamicin B1 (B1) is a potent readthrough inducer. Molecular dynamics simulations reveal the importance of ring I of B1 in establishing a ribosome configuration that permits pairing of a near-cognate complex at a PTC. B1 induced readthrough at all three nonsense codons in cultured cancer cells with TP53 (tumor protein p53) mutations, in cells from patients with nonsense mutations in the TPP1 (tripeptidyl peptidase 1), DMD (dystrophin), SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1), and COL7A1 (collagen type VII alpha 1 chain) genes, and in an in vivo tumor xenograft model. The B1 content of pharmaceutical gentamicin is highly variable and major gentamicins suppress the PTC readthrough activity of B1. Purified B1 provides a consistent and effective source of PTC readthrough activity to study the potential of nonsense suppression for treatment of rare genetic disorders.
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Benslimane, Nesrine, Federica Miressi, Camille Loret, Laurence Richard, Angélique Nizou, Ioanna Pyromali, Pierre-Antoine Faye, Frédéric Favreau, Fabrice Lejeune, and Anne-Sophie Lia. "Amlexanox: Readthrough Induction and Nonsense-Mediated mRNA Decay Inhibition in a Charcot–Marie–Tooth Model of hiPSCs-Derived Neuronal Cells Harboring a Nonsense Mutation in GDAP1 Gene." Pharmaceuticals 16, no. 7 (July 21, 2023): 1034. http://dx.doi.org/10.3390/ph16071034.
Full textAbstract:
Nonsense mutations are involved in multiple peripheral neuropathies. These mutations induce the presence of a premature termination codon (PTC) at the mRNA level. As a result, a dysfunctional or truncated protein is synthesized, or even absent linked to nonsense-mediated mRNA degradation (NMD) system activation. Readthrough molecules or NMD inhibitors could be innovative therapies in these hereditary neuropathies, particularly molecules harboring the dual activity as amlexanox. Charcot–Marie–Tooth (CMT) is the most common inherited pathology of the peripheral nervous system, affecting 1 in 2500 people worldwide. Nonsense mutations in the GDAP1 gene have been associated with a severe form of CMT, prompting us to investigate the effect of readthrough and NMD inhibitor molecules. Although not clearly defined, GDAP1 could be involved in mitochondrial functions, such as mitophagy. We focused on the homozygous c.581C>G (p.Ser194*) mutation inducing CMT2H using patient human induced pluripotent stem cell (hiPSC)-derived neuronal cells. Treatment during 20 h with 100 µM of amlexanox on this cell model stabilized GDAP1 mRNAs carrying UGA-PTC and induced a restoration of the mitochondrial morphology. These results highlight the potential of readthrough molecules associated to NMD inhibitors for the treatment of genetic alterations in CMT, opening the way for future investigations and a potential therapy.
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Schilff, Mirco, Yelena Sargsyan, Julia Hofhuis, and Sven Thoms. "Stop Codon Context-Specific Induction of Translational Readthrough." Biomolecules 11, no. 7 (July 9, 2021): 1006. http://dx.doi.org/10.3390/biom11071006.
Full textAbstract:
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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Inaoka, Takashi, Koji Kasai, and Kozo Ochi. "Construction of an In Vivo Nonsense Readthrough Assay System and Functional Analysis of Ribosomal Proteins S12, S4, and S5 in Bacillus subtilis." Journal of Bacteriology 183, no. 17 (September 1, 2001): 4958–63. http://dx.doi.org/10.1128/jb.183.17.4958-4963.2001.
Full textAbstract:
ABSTRACT To investigate the function of ribosomal proteins and translational factors in Bacillus subtilis, we developed an in vivo assay system to measure the level of nonsense readthrough by utilizing the LacZ-LacI system. Using the in vivo nonsense readthrough assay system which we developed, together with an in vitro poly(U)-directed cell-free translation assay system, we compared the processibility and translational accuracy of mutant ribosomes with those of the wild-type ribosome. Like Escherichia coli mutants, most S12 mutants exhibited lower frequencies of both UGA readthrough and missense error; the only exception was a mutant (in which Lys-56 was changed to Arg) which exhibited a threefold-higher frequency of readthrough than the wild-type strain. We also isolated several ribosomal ambiguity (ram) mutants from an S12 mutant. These ram mutants and the S12 mutant mentioned above (in which Lys-56 was changed to Arg) exhibited higher UGA readthrough levels. Thus, the mutation which altered Lys-56 to Arg resulted in aram phenotype in B. subtilis. The efficacy of our in vivo nonsense readthrough assay system was demonstrated in our investigation of the function of ribosomal proteins and translational factors.
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Yesmin, Farhana, Robiul H. Bhuiyan, Yuhsuke Ohmi, Yuki Ohkawa, Orie Tajima, Tetsuya Okajima, Keiko Furukawa, and Koichi Furukawa. "Aminoglycosides are efficient reagents to induce readthrough of premature termination codon in mutant B4GALNT1 genes found in families of hereditary spastic paraplegia." Journal of Biochemistry 168, no. 2 (May 27, 2020): 103–12. http://dx.doi.org/10.1093/jb/mvaa041.
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Abstract The readthrough of premature termination codon (PTC) by ribosome sometimes produces full-length proteins. We previously reported a readthrough of PTC of glycosyltransferase gene B4GALNT1 with hereditary spastic paraplegia (HSP). Here we featured the readthrough of B4GALNT1 of two mutants, M4 and M2 with PTC by immunoblotting and flow cytometry after transfection of B4GALNT1 cDNAs into cells. Immunoblotting showed a faint band of full-length mutant protein of M4 but not M2 at a similar position with that of wild-type B4GALNT1. AGC sequences at immediately before and after the PTC in M4 were critical for the readthrough. Treatment of cells transfected with mutant M4 cDNA with aminoglycosides resulted in increased readthrough of PTC. Furthermore, treatment of transfectants of mutant M2 cDNA with G418 also resulted in the induction of readthrough of PTC. Both M4 and M2 cDNA transfectants showed increased/induced bands in immunoblotting and GM2 expression in a dose-dependent manner of aminoglycosides. Results of mass spectrometry supported this effect. Here, we showed for the first time the induction and/or enhancement of the readthrough of PTCs of B4GALNT1 by aminoglycoside treatment, suggesting that aminoglycosides are efficient for patients with HSP caused by PTC of B4GALNT1, in which gradual neurological disorders emerged with aging.
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Carollo, Pietro Salvatore, Marco Tutone, Giulia Culletta, Ignazio Fiduccia, Federica Corrao, Ivana Pibiri, Aldo Di Leonardo, et al. "Investigating the Inhibition of FTSJ1, a Tryptophan tRNA-Specific 2′-O-Methyltransferase by NV TRIDs, as a Mechanism of Readthrough in Nonsense Mutated CFTR." International Journal of Molecular Sciences 24, no. 11 (June 1, 2023): 9609. http://dx.doi.org/10.3390/ijms24119609.
Full textAbstract:
Cystic Fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CFTR gene, coding for the CFTR chloride channel. About 10% of the CFTR gene mutations are “stop” mutations that generate a premature termination codon (PTC), thus synthesizing a truncated CFTR protein. A way to bypass PTC relies on ribosome readthrough, which is the ribosome’s capacity to skip a PTC, thus generating a full-length protein. “TRIDs” are molecules exerting ribosome readthrough; for some, the mechanism of action is still under debate. We investigate a possible mechanism of action (MOA) by which our recently synthesized TRIDs, namely NV848, NV914, and NV930, could exert their readthrough activity by in silico analysis and in vitro studies. Our results suggest a likely inhibition of FTSJ1, a tryptophan tRNA-specific 2′-O-methyltransferase.
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Li, Shan, Juan Li, Wenjing Shi, Ziyan Nie, Shasha Zhang, Fengdie Ma, Jun Hu, Jianjun Chen, Peiqiang Li, and Xiaodong Xie. "Pharmaceuticals Promoting Premature Termination Codon Readthrough: Progress in Development." Biomolecules 13, no. 6 (June 14, 2023): 988. http://dx.doi.org/10.3390/biom13060988.
Full textAbstract:
Around 11% of all known gene lesions causing human genetic diseases are nonsense mutations that introduce a premature stop codon (PTC) into the protein-coding gene sequence. Drug-induced PTC readthrough is a promising therapeutic strategy for treating hereditary diseases caused by nonsense mutations. To date, it has been found that more than 50 small-molecular compounds can promote PTC readthrough, known as translational readthrough-inducing drugs (TRIDs), and can be divided into two major categories: aminoglycosides and non-aminoglycosides. This review summarizes the pharmacodynamics and clinical application potential of the main TRIDs discovered so far, especially some newly discovered TRIDs in the past decade. The discovery of these TRIDs brings hope for treating nonsense mutations in various genetic diseases. Further research is still needed to deeply understand the mechanism of eukaryotic cell termination and drug-induced PTC readthrough so that patients can achieve the greatest benefit from the various TRID treatments.
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Du, Liutao, Robert Damoiseaux, Shareef Nahas, Kun Gao, Hailiang Hu, Julianne M. Pollard, Jimena Goldstine, et al. "Nonaminoglycoside compounds induce readthrough of nonsense mutations." Journal of Experimental Medicine 206, no. 10 (September 21, 2009): 2285–97. http://dx.doi.org/10.1084/jem.20081940.
Full textAbstract:
Large numbers of genetic disorders are caused by nonsense mutations for which compound-induced readthrough of premature termination codons (PTCs) might be exploited as a potential treatment strategy. We have successfully developed a sensitive and quantitative high-throughput screening (HTS) assay, protein transcription/translation (PTT)–enzyme-linked immunosorbent assay (ELISA), for identifying novel PTC-readthrough compounds using ataxia-telangiectasia (A-T) as a genetic disease model. This HTS PTT-ELISA assay is based on a coupled PTT that uses plasmid templates containing prototypic A-T mutated (ATM) mutations for HTS. The assay is luciferase independent. We screened ∼34,000 compounds and identified 12 low-molecular-mass nonaminoglycosides with potential PTC-readthrough activity. From these, two leading compounds consistently induced functional ATM protein in ATM-deficient cells containing disease-causing nonsense mutations, as demonstrated by direct measurement of ATM protein, restored ATM kinase activity, and colony survival assays for cellular radiosensitivity. The two compounds also demonstrated readthrough activity in mdx mouse myotube cells carrying a nonsense mutation and induced significant amounts of dystrophin protein.
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Mangkalaphiban, Kotchaphorn, Feng He, Robin Ganesan, Chan Wu, Richard Baker, and Allan Jacobson. "Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae." PLOS Genetics 17, no. 4 (April 20, 2021): e1009538. http://dx.doi.org/10.1371/journal.pgen.1009538.
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Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3’-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5’ of the stop codon, six nucleotides 3’ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3’-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3’-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 had milder effects. Additionally, we found low readthrough genes to have shorter 3’-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.
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McHugh, Daniel R., Calvin U. Cotton, and Craig A. Hodges. "Synergy between Readthrough and Nonsense Mediated Decay Inhibition in a Murine Model of Cystic Fibrosis Nonsense Mutations." International Journal of Molecular Sciences 22, no. 1 (December 31, 2020): 344. http://dx.doi.org/10.3390/ijms22010344.
Full textAbstract:
Many heritable genetic disorders arise from nonsense mutations, which generate premature termination codons (PTCs) in transcribed mRNA. PTCs ablate protein synthesis by prematurely terminating the translation of mutant mRNA, as well as reducing mutant mRNA quantity through targeted degradation by nonsense-mediated decay (NMD) mechanisms. Therapeutic strategies for nonsense mutations include facilitating ribosomal readthrough of the PTC and/or inhibiting NMD to restore protein function. However, the efficacy of combining readthrough agents and NMD inhibitors has not been thoroughly explored. In this study, we examined combinations of known NMD inhibitors and readthrough agents using functional analysis of the CFTR protein in primary cells from a mouse model carrying a G542X nonsense mutation in Cftr. We observed synergy between an inhibitor of the NMD component SMG-1 (SMG1i) and the readthrough agents G418, gentamicin, and paromomycin, but did not observe synergy with readthrough caused by amikacin, tobramycin, PTC124, escin, or amlexanox. These results indicate that treatment with NMD inhibitors can increase the quantity of functional protein following readthrough, and that combining NMD inhibitors and readthrough agents represents a potential therapeutic option for treating nonsense mutations.
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Chan, Clara S., Irwin Jungreis, and Manolis Kellis. "Heterologous Stop Codon Readthrough of Metazoan Readthrough Candidates in Yeast." PLoS ONE 8, no. 3 (March 27, 2013): e59450. http://dx.doi.org/10.1371/journal.pone.0059450.
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Belitsky, Boris R., Hyun-Jin Kim, and Abraham L. Sonenshein. "CcpA-Dependent Regulation of Bacillus subtilis Glutamate Dehydrogenase Gene Expression." Journal of Bacteriology 186, no. 11 (June 1, 2004): 3392–98. http://dx.doi.org/10.1128/jb.186.11.3392-3398.2004.
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ABSTRACT The Bacillus subtilis rocG gene, encoding catabolic glutamate dehydrogenase, was found to be subject to direct CcpA-dependent glucose repression. The effect of CcpA required the presence of both the HPr and Crh proteins. The primary CcpA binding site was identified by mutational analysis and DNase I footprinting. In the absence of inducers of the Roc pathway, rocG was still expressed at a low level due to readthrough transcription. CcpA-dependent repression of rocG readthrough transcription proved to contribute to the slow growth rate of B. subtilis cells in glucose-glutamate medium. Increased readthrough expression of rocG was shown to be partially responsible for the growth defect of ccpA strains in glucose-ammonium medium.
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Kosinski, Luke J., and Joanna Masel. "Readthrough Errors Purge Deleterious Cryptic Sequences, Facilitating the Birth of Coding Sequences." Molecular Biology and Evolution 37, no. 6 (February 26, 2020): 1761–74. http://dx.doi.org/10.1093/molbev/msaa046.
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Abstract De novo protein-coding innovations sometimes emerge from ancestrally noncoding DNA, despite the expectation that translating random sequences is overwhelmingly likely to be deleterious. The “preadapting selection” hypothesis claims that emergence is facilitated by prior, low-level translation of noncoding sequences via molecular errors. It predicts that selection on polypeptides translated only in error is strong enough to matter and is strongest when erroneous expression is high. To test this hypothesis, we examined noncoding sequences located downstream of stop codons (i.e., those potentially translated by readthrough errors) in Saccharomyces cerevisiae genes. We identified a class of “fragile” proteins under strong selection to reduce readthrough, which are unlikely substrates for co-option. Among the remainder, sequences showing evidence of readthrough translation, as assessed by ribosome profiling, encoded C-terminal extensions with higher intrinsic structural disorder, supporting the preadapting selection hypothesis. The cryptic sequences beyond the stop codon, rather than spillover effects from the regular C-termini, are primarily responsible for the higher disorder. Results are robust to controlling for the fact that stronger selection also reduces the length of C-terminal extensions. These findings indicate that selection acts on 3′ UTRs in Saccharomyces cerevisiae to purge potentially deleterious variants of cryptic polypeptides, acting more strongly in genes that experience more readthrough errors.
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Moss, Tom, Anne-Marie Larose, Keith Mitchelson, and Benoît Leblanc. "Readthrough enhancement and promoter occlusion on the ribosomal genes of Xenopus laevis." Biochemistry and Cell Biology 70, no. 5 (May 1, 1992): 324–31. http://dx.doi.org/10.1139/o92-050.
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An RNA polymerase I termination site is found just upstream of the ribosomal gene promoter in mammals and amphibia. It has been suggested that this termination site may actively enhance ribosomal transcription in a process known as readthrough enhancement or that it may simply prevent the disruption of initiation complexes or promoter occlusion. There is, however, a consensus of opinion that the terminator is important for efficient ribosomal transcription. Here we have quantitatively investigated the relative importance of readthrough enhancement and promoter occlusion on the transcription of the microinjected Xenopus laevis ribosomal gene. The results show that, in this system, promoter occlusion is limited and terminator mutations predominantly affect readthrough enhancement. The terminator is shown to be unnecessary for the enhancer activity of the rest of the ribosomal spacer. Model calculations suggest that readthrough enhancement could be explained by polymerase recycling and that it may be unnecessary to postulate a specific mechanism of polymerase handover.Key words: Xenopus, ribosomal, promoter, RNA polymerase I, rDNA.
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Dabrowski, Maciej, Zuzanna Bukowy-Bieryllo, Claire L. Jackson, and Ewa Zietkiewicz. "Properties of Non-Aminoglycoside Compounds Used to Stimulate Translational Readthrough of PTC Mutations in Primary Ciliary Dyskinesia." International Journal of Molecular Sciences 22, no. 9 (May 7, 2021): 4923. http://dx.doi.org/10.3390/ijms22094923.
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Primary ciliary dyskinesia (PCD) is a rare disease with autosomal recessive inheritance, caused mostly by bi-allelic gene mutations that impair motile cilia structure and function. Currently, there are no causal treatments for PCD. In many disease models, translational readthrough of premature termination codons (PTC-readthrough) induced by aminoglycosides has been proposed as an effective way of restoring functional protein expression and reducing disease symptoms. However, variable outcomes of pre-clinical trials and toxicity associated with long-term use of aminoglycosides prompt the search for other compounds that might overcome these problems. Because a high proportion of PCD-causing variants are nonsense mutations, readthrough therapies are an attractive option. We tested a group of chemical compounds with known PTC-readthrough potential (ataluren, azithromycin, tylosin, amlexanox, and the experimental compound TC007), collectively referred to as non-aminoglycosides (NAGs). We investigated their PTC-readthrough efficiency in six PTC mutations found in Polish PCD patients, in the context of cell and cilia health, and in comparison to the previously tested aminoglycosides. The NAGs did not compromise the viability of the primary nasal respiratory epithelial cells, and the ciliary beat frequency was retained, similar to what was observed for gentamicin. In HEK293 cells transfected with six PTC-containing inserts, the tested compounds stimulated PTC-readthrough but with lower efficiency than aminoglycosides. The study allowed us to select compounds with minimal negative impact on cell viability and function but still the potential to induce PTC-readthrough.
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Temaj, Gazmend, Pelin Telkoparan-Akillilar, Nexhibe Nuhii, Silvia Chichiarelli, Sarmistha Saha, and Luciano Saso. "Recoding of Nonsense Mutation as a Pharmacological Strategy." Biomedicines 11, no. 3 (February 22, 2023): 659. http://dx.doi.org/10.3390/biomedicines11030659.
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Approximately 11% of genetic human diseases are caused by nonsense mutations that introduce a premature termination codon (PTC) into the coding sequence. The PTC results in the production of a potentially harmful shortened polypeptide and activation of a nonsense-mediated decay (NMD) pathway. The NMD pathway reduces the burden of unproductive protein synthesis by lowering the level of PTC mRNA. There is an endogenous rescue mechanism that produces a full-length protein from a PTC mRNA. Nonsense suppression therapies aim to increase readthrough, suppress NMD, or are a combination of both strategies. Therefore, treatment with translational readthrough-inducing drugs (TRIDs) and NMD inhibitors may increase the effectiveness of PTC suppression. Here we discuss the mechanism of PTC readthrough and the development of novel approaches to PTC suppression. We also discuss the toxicity and bioavailability of therapeutics used to stimulate PTC readthrough.
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Gao, Shenglan, Ziying Lin, Chunyan Li, Yahong Wang, Lawei Yang, Bao’an Zou, Jie Chen, et al. "lncINS-IGF2 Promotes Cell Proliferation and Migration by Promoting G1/S Transition in Lung Cancer." Technology in Cancer Research & Treatment 18 (January 1, 2019): 153303381882302. http://dx.doi.org/10.1177/1533033818823029.
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Long noncoding RNAs are capable of regulating gene expression at multiple levels. These RNA molecules are also involved in a variety of physiological and pathological processes. Emerging data demonstrate that a series of differentially expressed long noncoding RNAs are implicated in tumorigenesis. In the present study, we used microarray analysis to identify long noncoding RNAs that are dysregulated in non-small-cell lung cancer when compared to normal lung tissues. Accordingly, we performed quantitative real-time polymerase chain reaction to analyze the levels of long noncoding RNA and the cis target gene. We further found the oncogene property of long noncoding RNA that long noncoding RNA downexpression inhibits non-small-cell lung cancer cells proliferation and migration based on 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide and colony formation assays and wound healing as well as transwell assays. The influence of long noncoding RNA on cell cycle of non-small-cell lung cancer cells is also analyzed by flow cytometry. Among the dysregulated long noncoding RNAs, we identified INS-IGF2 readthrough, transcript variant 1, noncoding RNA (NR_003512.3) is upregulated in non-small-cell lung cancer tissues, the cis gene of which is insulin-like growth factor 2 gene hinted by bioinformatics analysis. We also observed that downregulation of INS-IGF2 readthrough, transcript variant 1, noncoding RNA reduces insulin-like growth factor 2 messenger RNA expression. Furthermore, INS-IGF2 readthrough, transcript variant 1, noncoding RNA downregulation suppresses non-small-cell lung cancer cell proliferation and migration. This downregulation results in a concomitant inhibition of the G1/S transition in non-small-cell lung cancer cells. Our findings suggest that INS-IGF2 readthrough, transcript variant 1, noncoding RNA may be an oncogene involved in the development of lung cancer. Therefore, we speculate that INS-IGF2 readthrough, transcript variant 1, noncoding RNA represents a potential therapeutic target for lung cancer.
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Bralley, Patricia, and George H. Jones. "Organization and Expression of the Polynucleotide Phosphorylase Gene (pnp) of Streptomyces: Processing of pnp Transcripts in Streptomyces antibioticus." Journal of Bacteriology 186, no. 10 (May 15, 2004): 3160–72. http://dx.doi.org/10.1128/jb.186.10.3160-3172.2004.
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ABSTRACT We have examined the expression of pnp encoding the 3′-5′-exoribonuclease, polynucleotide phosphorylase, in Streptomyces antibioticus. We show that the rpsO-pnp operon is transcribed from at least two promoters, the first producing a readthrough transcript that includes both pnp and the gene for ribosomal protein S15 (rpsO) and a second, Ppnp, located in the rpsO-pnp intergenic region. Unlike the situation in Escherichia coli, where observation of the readthrough transcript requires mutants lacking RNase III, we detect readthrough transcripts in wild-type S. antibioticus mycelia. The Ppnp transcriptional start point was mapped by primer extension and confirmed by RNA ligase-mediated reverse transcription-PCR, a technique which discriminates between 5′ ends created by transcription initiation and those produced by posttranscriptional processing. Promoter probe analysis demonstrated the presence of a functional promoter in the intergenic region. The Ppnp sequence is similar to a group of promoters recognized by the extracytoplasmic function sigma factors, sigma-R and sigma-E. We note a number of other differences in rspO-pnp structure and function between S. antibioticus and E. coli. In E. coli, pnp autoregulation and cold shock adaptation are dependent upon RNase III cleavage of an rpsO-pnp intergenic hairpin. Computer modeling of the secondary structure of the S. antibioticus readthrough transcript predicts a stem-loop structure analogous to that in E. coli. However, our analysis suggests that while the readthrough transcript observed in S. antibioticus may be processed by an RNase III-like activity, transcripts originating from Ppnp are not. Furthermore, the S. antibioticus rpsO-pnp intergenic region contains two open reading frames. The larger of these, orfA, may be a pseudogene. The smaller open reading frame, orfX, also observed in Streptomyces coelicolor and Streptomyces avermitilis, may be translationally coupled to pnp and the gene downstream from pnp, a putative protease.
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Ranjitkar, S., M. Siri, J. Sun, G. Liu, and X. Tian. "117 Transcription readthrough in." Reproduction, Fertility and Development 35, no. 2 (December 5, 2022): 185–86. http://dx.doi.org/10.1071/rdv35n2ab117.
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Baradaran-Heravi, Alireza, Aruna D. Balgi, Carla Zimmerman, Kunho Choi, Fahimeh S. Shidmoossavee, Jason S. Tan, Célia Bergeaud, et al. "Novel small molecules potentiate premature termination codon readthrough by aminoglycosides." Nucleic Acids Research 44, no. 14 (July 12, 2016): 6583–98. http://dx.doi.org/10.1093/nar/gkw638.
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Morais, Pedro, Rui Zhang, and Yi-Tao Yu. "Therapeutic Nonsense Suppression Modalities: From Small Molecules to Nucleic Acid-Based Approaches." Biomedicines 12, no. 6 (June 10, 2024): 1284. http://dx.doi.org/10.3390/biomedicines12061284.
Full textAbstract:
Nonsense mutations are genetic mutations that create premature termination codons (PTCs), leading to truncated, defective proteins in diseases such as cystic fibrosis, neurofibromatosis type 1, Dravet syndrome, Hurler syndrome, Beta thalassemia, inherited bone marrow failure syndromes, Duchenne muscular dystrophy, and even cancer. These mutations can also trigger a cellular surveillance mechanism known as nonsense-mediated mRNA decay (NMD) that degrades the PTC-containing mRNA. The activation of NMD can attenuate the consequences of truncated, defective, and potentially toxic proteins in the cell. Since approximately 20% of all single-point mutations are disease-causing nonsense mutations, it is not surprising that this field has received significant attention, resulting in a remarkable advancement in recent years. In fact, since our last review on this topic, new examples of nonsense suppression approaches have been reported, namely new ways of promoting the translational readthrough of PTCs or inhibiting the NMD pathway. With this review, we update the state-of-the-art technologies in nonsense suppression, focusing on novel modalities with therapeutic potential, such as small molecules (readthrough agents, NMD inhibitors, and molecular glue degraders); antisense oligonucleotides; tRNA suppressors; ADAR-mediated RNA editing; targeted pseudouridylation; and gene/base editing. While these various modalities have significantly advanced in their development stage since our last review, each has advantages (e.g., ease of delivery and specificity) and disadvantages (manufacturing complexity and off-target effect potential), which we discuss here.
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Lowndes, N. F., P. Bushel, L. Mendelsohn, J. Wu, M. Y. Yen, and M. Allan. "A short, highly repetitive element in intron -1 of the human c-Ha-ras gene acts as a block to transcriptional readthrough by a viral promoter." Molecular and Cellular Biology 10, no. 9 (September 1990): 4990–95. http://dx.doi.org/10.1128/mcb.10.9.4990-4995.1990.
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We have identified a short, highly repetitive element within intron -1 of the human c-Ha-ras gene. This element was found to be transcribed in both orientations and to be homologous to heterogeneous nonpolyadenylated transcripts. The repetitive element blocked transcriptional readthrough from a strong upstream viral promoter but allowed unimpaired readthrough from the c-Has-ras promoter. We suggest that it may serve to prevent excessive transcription into the coding region of the gene under such circumstances as viral insertion.
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Ghelfi, Mikel D., Saleem Y. Bhat, Hong Li, and Barry S. Cooperman. "A High-Throughput Assay for In Vitro Determination of Release Factor-Dependent Peptide Release from a Pretermination Complex by Fluorescence Anisotropy—Application to Nonsense Suppressor Screening and Mechanistic Studies." Biomolecules 13, no. 2 (January 27, 2023): 242. http://dx.doi.org/10.3390/biom13020242.
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Premature termination codons (PTCs) account for ~12% of all human disease mutations. Translation readthrough-inducing drugs (TRIDs) are prominent among the several therapeutic approaches being used to overcome PTCs. Ataluren is the only TRID that has been approved for treating patients suffering from a PTC disease, Duchenne muscular dystrophy, but it gives variable readthrough results in cells isolated from patients suffering from other PTC diseases. We recently elucidated ataluren’s mechanism of action as a competitive inhibitor of release factor complex (RFC) catalysis of premature termination and identified ataluren’s binding sites on the ribosome responsible for such an inhibition. These results suggest the possibility of discovering new TRIDs, which would retain ataluren’s low toxicity while displaying greater potency and generality in stimulating readthrough via the inhibition of termination. Here we present a detailed description of a new in vitro plate reader assay that we are using both to screen small compound libraries for the inhibition of RFC-dependent peptide release and to better understand the influence of termination codon identity and sequence context on RFC activity.
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Yordanova, Martina M., Gary Loughran, John F. Atkins, and Pavel V. Baranov. "Stop codon readthrough contexts influence reporter expression differentially depending on the presence of an IRES." Wellcome Open Research 5 (January 31, 2022): 221. http://dx.doi.org/10.12688/wellcomeopenres.16231.3.
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Abstract Background: Previously we reported the discovery of stop codon readthrough in AMD1 mRNA followed by ribosome stalling at the end of a conserved Open Reading Frame (ORF) that we termed AMD1. To explain the severe suppression of reporters fused to AMD1 tail we proposed a mechanism invoking ribosome queueing. In the original study, we tested this hypothesis, by placing the reporter stop codon in the context of readthrough permissive sequences in a dual reporter vector with downstream reporter expression driven by the EMCV IRES. In accordance with our hypothesis, we observed a striking disproportional reduction of upstream reporter activity in response to increased readthrough levels. Methods: Here we employ dual luciferase assays, western blotting and RT-qPCR to explore the effects of test sequences downstream to the reporter stop codon on its expression in dual and monocistronic reporter vectors. Results: With the dual reporter system, the disproportionate reduction of upstream reporter activity is not specific to AMD1 tail and occurs as long as the readthrough stop codon context is present at the end of the reporter’s ORF. In a monocistronic vector without an IRES, the test sequences had distinct effects which were reflective of their properties e.g., AMD1 tail inhibitory effect. We further show by employing RT-qPCR that in the IRES vectors, the Fluc activity levels measured by the luciferase assay are an accurate proxy of RNA levels. Conclusions: While our findings provide little new information regarding the functional role of AMD1 tail, they raise caution for the use of viral IRES elements in expression vectors for studying mechanisms of mRNA translation. These findings may also be pertinent to the natural properties of readthrough permissive sequences and of IRES elements, though these require a separate investigation.
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Wong, Keit Men, Eike Wegener, Alireza Baradaran-Heravi, Brenda Huppke, Jutta Gärtner, and Peter Huppke. "Evaluation of Novel Enhancer Compounds in Gentamicin-Mediated Readthrough of Nonsense Mutations in Rett Syndrome." International Journal of Molecular Sciences 24, no. 14 (July 19, 2023): 11665. http://dx.doi.org/10.3390/ijms241411665.
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Rett syndrome (RTT), a severe X-linked neurodevelopmental disorder, is primarily caused by mutations in the methyl CpG binding protein 2 gene (MECP2). Over 35% RTT patients carry nonsense mutation in MECP2, making it a suitable candidate disease for nonsense suppression therapy. In our previous study, gentamicin was found to induce readthrough of MECP2 nonsense mutations with modest efficiency. Given the recent discovery of readthrough enhancers, CDX compounds, we herein evaluated the potentiation effect of CDX5-1, CDX5-288, and CDX6-180 on gentamicin-mediated readthrough efficiency in transfected HeLa cell lines bearing the four most common MECP2 nonsense mutations. We showed that all three CDX compounds potentiated gentamicin-mediated readthrough and increased full-length MeCP2 protein levels in cells expressing the R168X, R255X, R270X, and R294X nonsense mutations. Among all three CDX compounds, CDX5-288 was the most potent enhancer and enabled the use of reduced doses of gentamicin, thus mitigating the toxicity. Furthermore, we successfully demonstrated the upregulation of full-length Mecp2 protein expression in fibroblasts derived from Mecp2R255X/Y mice through combinatorial treatment. Taken together, findings demonstrate the feasibility of this combinatorial approach to nonsense suppression therapy for a subset of RTT patients.