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Alphaviruses are enveloped, single-stranded, positive-sense RNA viruses that often require transmission between arthropod and vertebrate hosts for their sustained propagation. Most alphaviruses encode an opal (UGA) termination codon in nonstructural protein 3 (nsP3) upstream of the viral polymerase, nsP4. The selective constraints underlying the conservation of the opal codon are poorly understood. Using primate and mosquito cells, we explored the role and selective pressure on the nsP3 opal codon through extensive mutational analysis in the prototype alphavirus, Sindbis virus (SINV). We found that the opal codon is highly favored over all other codons in primate cells under native 37°C growth conditions. However, this preference is diminished in mosquito and primate cells grown at a lower temperature. Thus, the primary determinant driving the selection of the opal stop codon is not host genetics but the passaging temperature. We show that the opal codon is preferred over amber and ochre termination codons because it results in the highest translational readthrough and polymerase production. However, substituting the opal codon with sense codons leads to excessive full-length polyprotein (P1234) production, which disrupts optimal nsP polyprotein processing, delays the switch from minus-strand to positive-strand RNA production, and significantly reduces SINV fitness at 37°C; this fitness defect is relieved at lower temperatures. A naturally occurring suppressor mutation unexpectedly compensates for a delayed transition from minus to genomic RNA production by also delaying the subsequent transition between genomic and sub-genomic RNA production. Our study reveals that the opal stop codon is the best solution for alphavirus replication at 37°C, producing enough nsP4 protein to maximize replication without disrupting nsP processing and RNA replication transitions needed for optimal fitness. Our study uncovers the intricate strategy dual-host alphaviruses use at a single codon to optimize fitness.
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FEM1B is a substrate-recognition component of the CRL2 E3 ubiquitin-protein ligase. This multi-protein complex targets specific proteins for ubiquitylation, which leads to their degradation. Here, we demonstrate the regulation of FEM1B expression by stop codon readthrough (SCR). In this process, translating ribosomes readthrough the stop codon of FEM1B to generate a C-terminally extended isoform that is highly unstable. A total of 81 nucleotides in the proximal 3'UTR of FEM1B constitute the necessary and sufficient cis-signal for SCR. Also, they encode the amino acid sequence responsible for the degradation of the SCR product. CRISPR-edited cells lacking this region, and therefore SCR of FEM1B, showed increased FEM1B expression. This in turn resulted in reduced expression of SLBP (a target of FEM1B-mediated degradation) and replication-dependent histones (target of SLBP for mRNA stability), causing cell cycle delay. Evolutionary analysis revealed that this phenomenon is specific to the genus Pan and Homo (Hominini). Overall, we show a relatively recently evolved SCR process that relieves the cell cycle from the negative regulation by FEM1B.
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Proteínas de Ciclo Celular , Ciclo Celular , Codón de Terminación , Humanos , Codón de Terminación/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/genética , Biosíntesis de Proteínas/genética , Animales , Regiones no Traducidas 3'/genética , Células HEK293 , Histonas/metabolismo , Histonas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Nucleares , Factores de Escisión y Poliadenilación de ARNmRESUMEN
The PTEN tumor suppressor is frequently targeted in tumors and patients with PTEN hamartoma tumor syndrome (PHTS) through nonsense mutations generating premature termination codons (PTC) that may cause the translation of truncated non-functional PTEN proteins. We have previously described a global analysis of the readthrough reconstitution of the protein translation and function of the human canonical PTEN isoform by aminoglycosides. Here, we report the efficient functional readthrough reconstitution of the PTEN translational isoform PTEN-L, which displays a minimal number of PTC in its specific N-terminal extension in association with disease. We illustrate the importance of the specific PTC and its nucleotide proximal sequence for optimal readthrough and show that the more frequent human PTEN PTC variants and their mouse PTEN PTC equivalents display similar patterns of readthrough efficiency. The heterogeneous readthrough response of the different PTEN PTC variants was independent of the length of the PTEN protein being reconstituted, and we found a correlation between the amount of PTEN protein being synthesized and the PTEN readthrough efficiency. Furthermore, combination of aminoglycosides and protein synthesis inducers increased the readthrough response of specific PTEN PTC. Our results provide insights with which to improve the functional reconstitution of human-disease-related PTC pathogenic variants from PTEN isoforms by increasing protein synthesis coupled to translational readthrough.
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Mutations in the TP53 tumor suppressor gene occur with high prevalence in a wide range of human tumors. A significant fraction of these mutations (around 10%) are nonsense mutations, creating a premature termination codon (PTC) that leads to the expression of truncated inactive p53 protein. Induction of translational readthrough across a PTC in nonsense mutant TP53 allows the production of full-length protein and potentially restoration of normal p53 function. Aminoglycoside antibiotics and a number of novel compounds have been shown to induce full-length p53 in tumor cells carrying various TP53 nonsense mutations. Full-length p53 protein generated by translational readthrough retains the capacity to transactivate p53 target genes and trigger tumor cell death. These findings raise hopes for efficient therapy of TP53 nonsense mutant tumors in the future.
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Codón sin Sentido , Neoplasias , Proteína p53 Supresora de Tumor , Humanos , Neoplasias/genética , Neoplasias/tratamiento farmacológico , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Aminoglicósidos/uso terapéutico , Aminoglicósidos/farmacologíaRESUMEN
Stop codon readthrough (SCR) is the process where translation continues beyond a stop codon on an mRNA. Here, we describe a strategy to enhance or induce SCR in a transcript-selective manner using a CRISPR-dCas13 system. Using specific guide RNAs, we target dCas13 to the region downstream of canonical stop codons of mammalian AGO1 and VEGFA mRNAs, known to exhibit natural SCR. Readthrough assays reveal enhanced SCR of these mRNAs (both exogenous and endogenous) caused by the dCas13-gRNA complexes. This effect is associated with ribosomal pausing, which has been reported for several SCR events. Our data show that CRISPR-dCas13 can also induce SCR across premature termination codons (PTCs) in the mRNAs of green fluorescent protein and TP53. We demonstrate the utility of this strategy in the induction of readthrough across the thalassemia-causing PTC in HBB mRNA and hereditary spherocytosis-causing PTC in SPTA1 mRNA. Thus, CRISPR-dCas13 can be programmed to enhance or induce SCR in a transcript-selective and stop codon-specific manner.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ARN Guía de Sistemas CRISPR-Cas , Animales , Codón de Terminación/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Codón sin Sentido/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Biosíntesis de Proteínas , Mamíferos/genética , Mamíferos/metabolismoRESUMEN
Rett syndrome (RTT) is a neurodevelopmental disorder resulting from genetic mutations in the methyl CpG binding protein 2 (MeCP2) gene. Specifically, around 35% of RTT patients harbor premature termination codons (PTCs) within the MeCP2 gene due to nonsense mutations. A promising therapeutic avenue for these individuals involves the use of aminoglycosides, which stimulate translational readthrough (TR) by causing stop codons to be interpreted as sense codons. However, the effectiveness of this treatment depends on several factors, including the type of stop codon and the surrounding nucleotides, collectively referred to as the stop codon context (SCC). Here, we develop a high-content reporter system to precisely measure TR efficiency at different SCCs, assess the recovery of the full-length MeCP2 protein, and evaluate its subcellular localization. We have conducted a comprehensive investigation into the intricate relationship between SCC characteristics and TR induction, examining a total of 14 pathogenic MeCP2 nonsense mutations with the aim to advance the prospects of personalized therapy for individuals with RTT. Our results demonstrate that TR induction can successfully restore full-length MeCP2 protein, albeit to varying degrees, contingent upon the SCC and the specific position of the PTC within the MeCP2 mRNA. TR induction can lead to the re-establishment of nuclear localization of MeCP2, indicating the potential restoration of protein functionality. In summary, our findings underscore the significance of SCC-specific approaches in the development of tailored therapies for RTT. By unraveling the relationship between SCC and TR therapy, we pave the way for personalized, individualized treatment strategies that hold promise for improving the lives of individuals affected by this debilitating neurodevelopmental disorder. KEY MESSAGES: The efficiency of readthrough induction at MeCP2 premature termination codons strongly depends on the stop codon context. The position of the premature termination codon on the transcript influences the readthrough inducibility. A new high-content dual reporter assay facilitates the measurement and prediction of readthrough efficiency of specific nucleotide stop contexts. Readthrough induction results in the recovery of full-length MeCP2 and its re-localization to the nucleus. MeCP2 requires only one of its annotated nuclear localization signals.
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Codón sin Sentido , Codón de Terminación , Proteína 2 de Unión a Metil-CpG , Síndrome de Rett , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Humanos , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células HEK293RESUMEN
Shwachman-Diamond syndrome (SDS) is characterized by neutropenia, exocrine pancreatic insufficiency and skeletal abnormalities. SDS bone marrow haematopoietic progenitors show increased apoptosis and impairment in granulocytic differentiation. Loss of Shwachman-Bodian-Diamond syndrome (SBDS) expression results in reduced eukaryotic 80S ribosome maturation. Biallelic mutations in the SBDS gene are found in ~90% of SDS patients, ~55% of whom carry the c.183-184TA>CT nonsense mutation. Several translational readthrough-inducing drugs aimed at suppressing nonsense mutations have been developed. One of these, ataluren, has received approval in Europe for the treatment of Duchenne muscular dystrophy. We previously showed that ataluren can restore full-length SBDS protein synthesis in SDS-derived bone marrow cells. Here, we extend our preclinical study to assess the functional restoration of SBDS capabilities in vitro and ex vivo. Ataluren improved 80S ribosome assembly and total protein synthesis in SDS-derived cells, restored myelopoiesis in myeloid progenitors, improved neutrophil chemotaxis in vitro and reduced neutrophil dysplastic markers ex vivo. Ataluren also restored full-length SBDS synthesis in primary osteoblasts, suggesting that its beneficial role may go beyond the myeloid compartment. Altogether, our results strengthened the rationale for a Phase I/II clinical trial of ataluren in SDS patients who harbour the nonsense mutation.
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Enfermedades de la Médula Ósea , Insuficiencia Pancreática Exocrina , Lipomatosis , Humanos , Síndrome de Shwachman-Diamond , Proteína p53 Supresora de Tumor/genética , Lipomatosis/genética , Codón sin Sentido , Mielopoyesis , Neutrófilos/metabolismo , Quimiotaxis , Enfermedades de la Médula Ósea/genética , Enfermedades de la Médula Ósea/terapia , Insuficiencia Pancreática Exocrina/genética , Ribosomas/metabolismoRESUMEN
BACKGROUND: Coagulation factor V (FV) deficiency is a rare bleeding disorder that is usually managed with fresh-frozen plasma. Patients with nonsense mutations may respond to treatment with readthrough agents. OBJECTIVES: To investigate whether the F5 p.Arg1161Ter mutation, causing severe FV deficiency in several patients, would be amenable to readthrough therapy. METHODS: F5 mRNA and protein expression were evaluated in a F5 p.Arg1161Ter-homozygous patient. Five readthrough agents with different mechanisms of action, i.e. G418, ELX-02, PTC-124, 2,6-diaminopurine (2,6-DAP), and Amlexanox, were tested in in vitro and ex vivo models of the mutation. RESULTS: The F5 p.Arg1161Ter-homozygous patient showed residual F5 mRNA and functional platelet FV, indicating detectable levels of natural readthrough. COS-1 cells transfected with the FV-Arg1161Ter cDNA expressed 0.7% FV activity compared to wild-type. Treatment with 0-500 µM G418, ELX-02, and 2,6-DAP dose-dependently increased FV activity up to 7.0-fold, 3.1-fold, and 10.8-fold, respectively, whereas PTC-124 and Amlexanox (alone or in combination) were ineffective. These findings were confirmed by thrombin generation assays in FV-depleted plasma reconstituted with conditioned media of treated cells. All compounds except ELX-02 showed some degree of cytotoxicity. Ex vivo differentiated megakaryocytes of the F5 p.Arg1161Ter-homozygous patient, which were negative at FV immunostaining, turned positive after treatment with all 5 readthrough agents. Notably, they were also able to internalize mutant FV rescued with G418 or 2,6-DAP, which would be required to maintain the crucial platelet FV pool in vivo. CONCLUSION: These findings provide in vitro and ex vivo proof-of-principle for readthrough-mediated rescue of the F5 p.Arg1161Ter mutation.
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Codón sin Sentido , Deficiencia del Factor V , Humanos , Factor V/genética , Factor V/metabolismo , Deficiencia del Factor V/tratamiento farmacológico , Deficiencia del Factor V/genética , Aminopiridinas , MutaciónRESUMEN
Nonsense mutations generating premature termination codons (PTCs) in various genes are frequently associated with somatic cancer and hereditary human diseases since PTCs commonly generate truncated proteins with defective or altered function. Induced translational readthrough during protein biosynthesis facilitates the incorporation of an amino acid at the position of a PTC, allowing the synthesis of a complete protein. This may evade the pathological effect of the PTC mutation and provide new therapeutic opportunities. Several protein tyrosine phosphatases (PTPs) genes are targeted by PTC in human disease, the tumor suppressor PTEN being the more prominent paradigm. Here, using PTEN and laforin as examples, two PTPs from the dual-specificity phosphatase subfamily, we describe methodologies to analyze in silico the distribution and frequency of pathogenic PTC in PTP genes. We also summarize laboratory protocols and technical notes to study the induced translational readthrough reconstitution of the synthesis of PTP targeted by PTC in association with disease in cellular models.
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Codón sin Sentido , Proteínas Tirosina Fosfatasas , Humanos , Mutación , Proteínas Tirosina Fosfatasas/genética , Fosfatasas de Especificidad Dual , Biosíntesis de ProteínasRESUMEN
Chikungunya virus (CHIKV) is an alphavirus, transmitted by Aedes species mosquitoes. The CHIKV single-stranded positive-sense RNA genome contains two open reading frames, coding for the non-structural (nsP) and structural proteins of the virus. The non-structural polyprotein precursor is proteolytically cleaved to generate nsP1-4. Intriguingly, most isolates of CHIKV (and other alphaviruses) possess an opal stop codon close to the 3' end of the nsP3 coding sequence and translational readthrough is necessary to produce full-length nsP3 and the nsP4 RNA polymerase. Here we investigate the role of this stop codon by replacing the arginine codon with each of the three stop codons in the context of both a subgenomic replicon and infectious CHIKV. Both opal and amber stop codons were tolerated in mammalian cells, but the ochre was not. In mosquito cells all three stop codons were tolerated. Using SHAPE analysis we interrogated the structure of a putative stem loop 3' of the stop codon and used mutagenesis to probe the importance of a short base-paired region at the base of this structure. Our data reveal that this stem is not required for stop codon translational readthrough, and we conclude that other factors must facilitate this process to permit productive CHIKV replication.
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Aedes , Fiebre Chikungunya , Virus Chikungunya , Animales , Virus Chikungunya/genética , Codón de Terminación/genética , Codón de Terminación/metabolismo , Fiebre Chikungunya/genética , Proteínas no Estructurales Virales/genética , Replicación Viral/genética , Mamíferos/genética , Mamíferos/metabolismoRESUMEN
Nonsense mutations cause several genetic diseases such as cystic fibrosis, Duchenne muscular dystrophy, ß-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 ß2M) 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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Genes Esenciales , Proteína p53 Supresora de Tumor , Codón de Terminación , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Biosíntesis de Proteínas , Codón sin SentidoRESUMEN
During translation, a stop codon on the mRNA signals the ribosomes to terminate the process. In certain mRNAs, the termination fails due to the recoding of the canonical stop codon, and ribosomes continue translation to generate C-terminally extended protein. This process, termed stop codon readthrough (SCR), regulates several cellular functions. SCR is driven by elements/factors that act immediately downstream of the stop codon. Here, we have analysed the process of SCR using a simple mathematical model to investigate how the kinetics of translating ribosomes influences the efficiency of SCR. Surprisingly, the analysis revealed that the rate of translation inversely regulates the efficiency of SCR. We tested this prediction experimentally in mammalian AGO1 and MTCH2 mRNAs. Reduction in translation either globally by harringtonine or locally by rare codons caused an increase in the efficiency of SCR. Thus, our study has revealed a hitherto unknown mode of regulation of SCR.
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Codón de Terminación , Biosíntesis de Proteínas , ARN Mensajero , Ribosomas , Codón de Terminación/genética , Codón de Terminación/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Humanos , Células HEK293 , Factores Eucarióticos de Iniciación/genética , Factores Eucarióticos de Iniciación/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismoRESUMEN
Termination codon readthrough (TCR) is a process in which ribosomes continue to translate an mRNA beyond a stop codon generating a C-terminally extended protein isoform. Here, we demonstrate TCR in mammalian NNAT mRNA, which encodes NNAT, a proteolipid important for neuronal differentiation. This is a programmed event driven by cis-acting RNA sequences present immediately upstream and downstream of the canonical stop codon and is negatively regulated by NONO, an RNA-binding protein known to promote neuronal differentiation. Unlike the canonical isoform NNAT, we determined that the TCR product (NNATx) does not show detectable interaction with the sarco/endoplasmic reticulum Ca2+-ATPase isoform 2 Ca2+ pump, cannot increase cytoplasmic Ca2+ levels, and therefore does not enhance neuronal differentiation in Neuro-2a cells. Additionally, an antisense oligonucleotide that targets a region downstream of the canonical stop codon reduced TCR of NNAT and enhanced the differentiation of Neuro-2a cells to cholinergic neurons. Furthermore, NNATx-deficient Neuro-2a cells, generated using CRISPR-Cas9, showed increased cytoplasmic Ca2+ levels and enhanced neuronal differentiation. Overall, these results demonstrate regulation of neuronal differentiation by TCR of NNAT. Importantly, this process can be modulated using a synthetic antisense oligonucleotide.
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Calcio , Neuronas , Biosíntesis de Proteínas , Animales , Calcio/metabolismo , Diferenciación Celular , Codón de Terminación , Mamíferos/metabolismo , Oligonucleótidos Antisentido/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Neuronas/citologíaRESUMEN
Nonsense mutations occur within the open-reading frame of a gene resulting in a premature termination codon (PTC). PTC-containing mRNAs can either be degeraded or cause premature translation termination producing a truncated protein that can be either nonfunctional or toxic. Translational readthrough inducing drugs (TRIDs) are small molecules that are able to induce readthrough, resulting in the restoration of full-length protein expression. The re-expressed proteins usually harbor a missense change. The effciency of individual TRIDs is variable and varies between different genes and even different nonsense mutations in the same gene. This review summarizes factors, including the sequences located upstream and downstream the disease-causing mutation and the type of PTC, affecting the translational readthrough process by modulating the type of amino acid insertion and the efficiency of the process during readthrough following TRIDs treatments.
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Codón sin Sentido , Biosíntesis de Proteínas , Codón sin Sentido/genética , Biosíntesis de Proteínas/genética , Aminoácidos , ARN Mensajero/genéticaRESUMEN
Congenital aniridia is a rare, pan-ocular disease causing severe sight loss, with only symptomatic intervention offered to patients. Approximately 40% of aniridia patients present with heterozygous nonsense variants in PAX6, resulting in haploinsufficiency. Translational readthrough-inducing drugs (TRIDs) have the ability to weaken the recognition of in-frame premature termination codons (PTCs), permitting full-length protein to be translated. We established induced pluripotent stem cell (iPSC)-derived 3D optic cups and 2D limbal epithelial stem cell (LESC) models from two aniridia patients with prevalent PAX6 nonsense mutations. Both in vitro models show reduced PAX6 protein levels, mimicking the disease. The repurposed TRIDs amlexanox and 2,6-diaminopurine (DAP) and the positive control compounds ataluren and G418 were tested for their efficiency. Amlexanox was identified as the most promising TRID, increasing full-length PAX6 levels in both models and rescuing the disease phenotype through normalization of VSX2 and cell proliferation in the optic cups and reduction of ABCG2 protein and SOX10 expression in LESCs. This study highlights the significance of patient iPSC-derived cells as a new model system for aniridia and proposes amlexanox as a new putative treatment for nonsense-mediated aniridia.
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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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Codón sin Sentido , Biosíntesis de Proteínas , Humanos , Codón sin Sentido/genética , Aminoglicósidos/farmacología , Aminoglicósidos/uso terapéutico , Antibacterianos/farmacología , Preparaciones FarmacéuticasRESUMEN
The TP53 and PTEN tumour suppressor genes are inactivated by nonsense mutations in a significant fraction of human tumours. TP53 nonsense mutatant tumours account for approximately one million new cancer cases per year worldwide. We have screened chemical libraries with the aim of identifying compounds that induce translational readthrough and expression of full-length p53 protein in cells with nonsense mutation in this gene. Here we describe two novel compounds with readthrough activity, either alone or in combination with other known readthrough-promoting substances. Both compounds induced levels of full-length p53 in cells carrying R213X nonsense mutant TP53. Compound C47 showed synergy with the aminoglycoside antibiotic and known readthrough inducer G418, whereas compound C61 synergized with eukaryotic release factor 3 (eRF3) degraders CC-885 and CC-90009. C47 alone showed potent induction of full-length PTEN protein in cells with different PTEN nonsense mutations. These results may facilitate further development of novel targeted cancer therapy by pharmacological induction of translational readthrough.
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Aminoglicósidos , Neoplasias , Humanos , Aminoglicósidos/farmacología , Codón sin Sentido , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Antibacterianos/farmacología , Inhibidores de la Síntesis de la ProteínaRESUMEN
Ten percent of cystic fibrosis (CF) patients carry a premature termination codon (PTC); no mutation-specific therapies exist for these individuals. ELX-02, a synthetic aminoglycoside, suppresses translation termination at PTCs (i.e., readthrough) by promoting the insertion of an amino acid at the PTC and restoring expression of full-length CFTR protein. The identity of amino acids inserted at PTCs affects the processing and function of the resulting full-length CFTR protein. We examined readthrough of the rare G550X-CFTR nonsense mutation due to its unique properties. We found that forskolin-induced swelling in G550X patient-derived intestinal organoids (PDOs) was significantly higher than in G542X PDOs (both UGA PTCs) with ELX-02 treatment, indicating greater CFTR function from the G550X allele. Using mass spectrometry, we identified tryptophan as the sole amino acid inserted in the G550X position during ELX-02- or G418-mediated readthrough, which differs from the three amino acids (cysteine, arginine, and tryptophan) inserted in the G542X position after treatment with G418. Compared with wild-type CFTR, Fischer rat thyroid (FRT) cells expressing the G550W-CFTR variant protein exhibited significantly increased forskolin-activated Cl- conductance, and G550W-CFTR channels showed increased PKA sensitivity and open probability. After treatment with ELX-02 and CFTR correctors, CFTR function rescued from the G550X allele in FRTs reached 20-40% of the wild-type level. These results suggest that readthrough of G550X produces greater CFTR function because of gain-of-function properties of the CFTR readthrough product that stem from its location in the signature LSGGQ motif found in ATP-binding cassette (ABC) transporters. G550X may be a particularly sensitive target for translational readthrough therapy.NEW & NOTEWORTHY We found that forskolin-induced swelling in G550X-CFTR patient-derived intestinal organoids (PDOs) was significantly higher than in G542X-CFTR PDOs after treatment with ELX-02. Tryptophan (W) was the sole amino acid inserted in the G550X position after readthrough. Resulting G550W-CFTR protein exhibited supernormal CFTR activity, PKA sensitivity, and open probability. These results show that aminoglycoside-induced readthrough of G550X produces greater CFTR function because of the gain-of-function properties of the CFTR readthrough product.
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Aminoglicósidos , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Ratas , Animales , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Aminoglicósidos/farmacología , Triptófano/genética , Colforsina/farmacología , Codón sin Sentido , Antibacterianos , Inhibidores de la Síntesis de la Proteína , Aminoácidos/genética , Ratas Endogámicas F344RESUMEN
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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Codón sin Sentido , Enfermedades Genéticas Congénitas , Extensión de la Cadena Peptídica de Translación , Medicina de Precisión , Enfermedades Raras , Supresión Genética , Animales , Humanos , Neoplasias de la Mama/genética , Neoplasias de la Mama/terapia , Codón sin Sentido/genética , Fibrosis Quística/genética , Fibrosis Quística/terapia , Epidermólisis Ampollosa/genética , Epidermólisis Ampollosa/terapia , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/terapia , Nefritis Hereditaria/genética , Nefritis Hereditaria/terapia , Degradación de ARNm Mediada por Codón sin Sentido , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Medicina de Precisión/métodos , Medicina de Precisión/tendencias , Enfermedades Raras/genética , Enfermedades Raras/terapia , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/terapia , Síndrome de Shwachman-Diamond/genética , Síndrome de Shwachman-Diamond/terapia , Supresión Genética/efectos de los fármacos , Supresión Genética/genética , Terminación de la Cadena Péptídica Traduccional/efectos de los fármacos , Aminoglicósidos/farmacologíaRESUMEN
Human Usher syndrome (USH) is the most common form of hereditary combined deaf-blindness. USH is a complex genetic disorder, and the pathomechanisms underlying the disease are far from being understood, especially in the eye and retina. The USH1C gene encodes the scaffold protein harmonin which organizes protein networks due to binary interactions with other proteins, such as all USH proteins. Interestingly, only the retina and inner ear show a disease-related phenotype, although USH1C/harmonin is almost ubiquitously expressed in the human body and upregulated in colorectal cancer. We show that harmonin binds to ß-catenin, the key effector of the canonical Wnt (cWnt) signaling pathway. We also demonstrate the interaction of the scaffold protein USH1C/harmonin with the stabilized acetylated ß-catenin, especially in nuclei. In HEK293T cells, overexpression of USH1C/harmonin significantly reduced cWnt signaling, but a USH1C-R31* mutated form did not. Concordantly, we observed an increase in cWnt signaling in dermal fibroblasts derived from an USH1C R31*/R80Pfs*69 patient compared with healthy donor cells. RNAseq analysis reveals that both the expression of genes related to the cWnt signaling pathway and cWnt target genes were significantly altered in USH1C patient-derived fibroblasts compared to healthy donor cells. Finally, we show that the altered cWnt signaling was reverted in USH1C patient fibroblast cells by the application of Ataluren, a small molecule suitable to induce translational read-through of nonsense mutations, hereby restoring some USH1C expression. Our results demonstrate a cWnt signaling phenotype in USH establishing USH1C/harmonin as a suppressor of the cWnt/ß-catenin pathway.