RESUMEN
The YgjD/Kae1 family (COG0533) has been on the top-10 list of universally conserved proteins of unknown function for over 5 years. It has been linked to DNA maintenance in bacteria and mitochondria and transcription regulation and telomere homeostasis in eukaryotes, but its actual function has never been found. Based on a comparative genomic and structural analysis, we predicted this family was involved in the biosynthesis of N(6)-threonylcarbamoyl adenosine, a universal modification found at position 37 of tRNAs decoding ANN codons. This was confirmed as a yeast mutant lacking Kae1 is devoid of t(6)A. t(6)A(-) strains were also used to reveal that t(6)A has a critical role in initiation codon restriction to AUG and in restricting frameshifting at tandem ANN codons. We also showed that YaeZ, a YgjD paralog, is required for YgjD function in vivo in bacteria. This work lays the foundation for understanding the pleiotropic role of this universal protein family.
Asunto(s)
Adenosina/análogos & derivados , Metaloendopeptidasas/metabolismo , Proteínas Mitocondriales/metabolismo , ARN de Transferencia/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Adenosina/metabolismo , Prueba de Complementación Genética , Metaloendopeptidasas/genética , Proteínas Mitocondriales/genética , Complejos Multiproteicos , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Prion proteins are found in mammals and yeast, and can transmit diseases and encode heritable phenotypic traits. In Saccharomyces cerevisiae, eRF3, Rnq1, Ure2 and Swil are functional proteins with a soluble conformation that can switch to a non-functional, amyloid conformation denoted as [PSI+], [PIN+], [URE3] and [SWI+], respectively. The prion [PSI+] corresponds to an aggregated conformation of the translational release factor eRF3, which suppresses nonsense codons. [PSI+] modifies cellular fitness and induces several phenotypes according to the genetic background. An elegant series of studies has demonstrated that several [PSI+]-induced phenotypes occur as a consequence of decreased translational termination efficiency. However, the genes whose expression levels are controlled by [PSI+] remain largely unknown. Here, we show that [PSI+] enhances expression of antizyme, a negative regulator of cellular polyamines, by modulating the +1 frameshifting required for its expression. Our study also demonstrates that [PSI+] greatly affects cellular polyamines in yeast. We show that modification of the cellular content of polyamines by the prion accounts for half of the [PSI+]-induced phenotypes. Antizyme is the first protein to be described for which expression of its functional form is stimulated by [PSI+].
Asunto(s)
Epigénesis Genética , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/metabolismo , Poliaminas/metabolismo , Priones/química , Priones/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Espacio Intracelular/metabolismo , Modelos Biológicos , Fenotipo , Saccharomyces cerevisiae/citologíaRESUMEN
The efficiency of translation termination depends on the nature of the stop codon and the surrounding nucleotides. Some molecules, such as aminoglycoside antibiotics (gentamicin), decrease termination efficiency and are currently being evaluated for diseases caused by premature termination codons. However, the readthrough response to treatment is highly variable and little is known about the rules governing readthrough level and response to aminoglycosides. In this study, we carried out in-depth statistical analysis on a very large set of nonsense mutations to decipher the elements of nucleotide context responsible for modulating readthrough levels and gentamicin response. We quantified readthrough for 66 sequences containing a stop codon, in the presence and absence of gentamicin, in cultured mammalian cells. We demonstrated that the efficiency of readthrough after treatment is determined by the complex interplay between the stop codon and a larger sequence context. There was a strong positive correlation between basal and induced readthrough levels, and a weak negative correlation between basal readthrough level and gentamicin response (i.e. the factor of increase from basal to induced readthrough levels). The identity of the stop codon did not affect the response to gentamicin treatment. In agreement with a previous report, we confirm that the presence of a cytosine in +4 position promotes higher basal and gentamicin-induced readthrough than other nucleotides. We highlight for the first time that the presence of a uracil residue immediately upstream from the stop codon is a major determinant of the response to gentamicin. Moreover, this effect was mediated by the nucleotide itself, rather than by the amino-acid or tRNA corresponding to the -1 codon. Finally, we point out that a uracil at this position associated with a cytosine at +4 results in an optimal gentamicin-induced readthrough, which is the therapeutically relevant variable.
Asunto(s)
Aminoácidos/genética , Codón sin Sentido , Gentamicinas , Terminación de la Cadena Péptídica Traduccional , ARN de Transferencia/genética , Células Cultivadas , Codón sin Sentido/efectos de los fármacos , Codón de Terminación/efectos de los fármacos , Citosina , Gentamicinas/farmacología , Humanos , Terminación de la Cadena Péptídica Traduccional/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , UraciloRESUMEN
Mutation-based treatments are a new development in genetic medicine, in which the nature of the mutation dictates the therapeutic strategy. Interest has recently focused on diseases caused by premature termination codons (PTCs). Drugs inducing the readthrough of these PTCs restore the production of a full-length protein. In this study, we explored the possibility of using aminoglycoside antibiotics to induce the production of a full-length functional p53 protein from a gene carrying a PTC. We identified a human cancer cell line containing a PTC, for which high levels of readthrough were obtained in the presence of aminoglycosides. Using these cells, we demonstrated that aminoglycoside treatment stabilized the mutant mRNA, which would otherwise have been degraded by non-sense-mediated decay, resulting in the production of a functional full-length p53 protein. Finally, we showed that aminoglycoside treatment decreased the viability of cancer cells specifically in the presence of nonsense-mutated p53 gene. These results open possibilities of developing promising treatments of cancers linked with non-sense mutations in tumor suppressor genes. They show that molecules designed to induce stop-codon readthrough can be used to inhibit tumor growth and offer a rational basis for developing new personalized strategies that could diversify the existing arsenal of cancer therapies.
Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , Antineoplásicos/farmacología , Codón sin Sentido , Genes p53 , Amicacina/farmacología , Animales , Apoptosis , Línea Celular , Línea Celular Tumoral , Gentamicinas/farmacología , Humanos , Ratones , ARN Mensajero/metabolismo , Activación Transcripcional , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The epigenetic factor [PSI+] in the yeast Saccharomyces cerevisiae is due to the prion form of Sup35p. The N-terminal domain of Sup35p (N), alone or together with the middle-domain (NM), assembles in vitro into fibrils that induce [PSI+] when introduced into yeast cells. The Sup35p C-terminal domain (C), involved in translation termination, is essential for growth. The involvement of Sup35p C-terminal domain into [PSI+] propagation is subject to debate. We previously showed that mutation of threonine 341 within Sup35p C-domain affects translation termination efficiency. Here, we demonstrate that mutating threonine 341 to aspartate or alanine results in synthetic lethality with [PSI+] and weakening of [PSI+] respectively. The corresponding Sup35D and Sup35A proteins assemble into wild-type like fibrils in vitro, but with a slower elongation rate. Moreover, cross-seeding between Sup35p and Sup35A is inefficient both in vivo and in vitro, suggesting that the point mutation alters the structural properties of Sup35p within the fibrils. Thus, Sup35p C-terminal domain modulates [PSI+] prion propagation, possibly through a functional interaction with the N and/or M domains of the protein. Our results clearly demonstrate that Sup35p C-terminal domain plays a critical role in prion propagation and provide new insights into the mechanism of prion conversion.
Asunto(s)
Mutación Missense , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Priones/genética , Priones/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sustitución de Aminoácidos , Viabilidad Microbiana , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Desnaturalización Proteica , Multimerización de ProteínaRESUMEN
Ten percent of human hereditary diseases are linked to nonsense mutations (premature termination codon). These mutations lead to premature translation termination, trigger the synthesis of a truncated protein and possibly lead to mRNA degradation by the NMD pathway (nonsense mediated mRNA decay). For the past ten years, therapeutic strategies have emerged which attempt to use molecules that facilitate tRNA incorporation at premature stop codon (readthrough), thus allowing for the synthesis of a full length protein. Molecules currently used for this approach are mostly aminoglycoside antibiotics (gentamicin, amikacin ) that bind the decoding center of the ribosome. This therapeutic approach has been studied for various genetic diseases including Duchenne muscular dystrophy (DMD) and cystic fibrosis. The feasibility of this approach depends on induced readthrough level, mRNA quantity, re-expressed protein functionality and characteristics of each disease.
Asunto(s)
Codón sin Sentido/fisiología , Enfermedades Genéticas Congénitas/terapia , Terapia Genética/métodos , Factores de Terminación de Péptidos/fisiología , Alelos , Codón sin Sentido/genética , Enfermedades Genéticas Congénitas/genética , Humanos , Modelos Biológicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida/métodos , Terminación de la Cadena Péptídica Traduccional/efectos de los fármacos , Terminación de la Cadena Péptídica Traduccional/genética , Factores de Terminación de Péptidos/genética , Especificidad por Sustrato/genética , Transcripción Genética/genética , Transcripción Genética/fisiologíaRESUMEN
Translation termination in eukaryotes is completed by two interacting factors eRF1 and eRF3. In Saccharomyces cerevisiae, these proteins are encoded by the genes SUP45 and SUP35, respectively. The eRF1 protein interacts directly with the stop codon at the ribosomal A-site, whereas eRF3-a GTPase protein-probably acts as a proofreading factor, coupling stop codon recognition to polypeptide chain release. We performed random PCR mutagenesis of SUP45 and screened the library for mutations resulting in increased eRF1 activity. These mutations led to the identification of two new pockets in domain 1 (P1 and P2) involved in the regulation of eRF1 activity. Furthermore, we identified novel mutations located in domains 2 and 3, which confer stop codon specificity to eRF1. Our findings are consistent with the model of a closed-active conformation of eRF1 and shed light on two new functional regions of the protein.
Asunto(s)
Terminación de la Cadena Péptídica Traduccional , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia Conservada , Eliminación de Gen , Prueba de Complementación Genética , Modelos Moleculares , Mutación , Factores de Terminación de Péptidos/metabolismo , Fenotipo , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Important regions of rRNA are rich in nucleotide modifications that can have strong effects on ribosome biogenesis and translation efficiency. Here, we examine the influence of pseudouridylation and 2'-O-methylation on translation accuracy in yeast, by deleting the corresponding guide snoRNAs. The regions analyzed were: the decoding centre (eight modifications), and two intersubunit bridge domains-the A-site finger and Helix 69 (six and five modifications). Results show that a number of modifications influence accuracy with effects ranging from 0.3- to 2.4-fold of wild-type activity. Blocking subsets of modifications, especially from the decoding region, impairs stop codon termination and reading frame maintenance. Unexpectedly, several Helix 69 mutants possess ribosomes with increased fidelity. Consistent with strong positional and synergistic effects is the finding that single deletions can have a more pronounced phenotype than multiple deficiencies in the same region. Altogether, the results demonstrate that rRNA modifications have significant roles in translation accuracy.
Asunto(s)
Biosíntesis de Proteínas , ARN Ribosómico/química , Saccharomyces cerevisiae/genética , Secuencia de Bases , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Nucleótidos/química , ARN de Hongos/químicaRESUMEN
Errors occur randomly and at low frequency during the translation of mRNA. However, such errors may also be programmed by the sequence and structure of the mRNA. These programmed events are called 'recoding' and are found mostly in viruses, in which they are usually essential for viral replication. Translational errors at a stop codon may also be induced by drugs, raising the possibility of developing new treatment protocols for genetic diseases on the basis of nonsense mutations. Many studies have been carried out, but the molecular mechanisms governing these events remain largely unknown. Studies on the yeast Saccharomyces cerevisiae have contributed to characterization of the HIV-1 frameshifting site and have demonstrated that frameshifting is conserved from yeast to humans. Yeast has also proved a particularly useful model organism for deciphering the mechanisms of translation termination in eukaryotes and identifying the factors required to obtain a high level of natural suppression. These findings open up new possibilities for large-scale screening in yeast to identify new drugs for blocking HIV replication by inhibiting frameshifting or restoring production of the full-length protein from a gene inactivated by a premature termination codon. We explore these two aspects of the contribution of yeast studies to human medicine in this review.
Asunto(s)
Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sistema de Lectura Ribosómico/efectos de los fármacos , Humanos , Terminación de la Cadena Péptídica Traduccional/efectos de los fármacos , Supresión GenéticaRESUMEN
In universal-code eukaryotes, a single-translation termination factor, eukaryote class-1 polypeptide release factor (eRF1), decodes the three stop codons: UAA, UAG, and UGA. In some ciliates, like Stylonychia and Paramecium, eRF1s exhibit UGA-only decoding specificity, whereas UAG and UAA are reassigned as sense codons. Because variant-code ciliates may have evolved from universal-code ancestor(s), structural features should exist in ciliate eRF1s that restrict their stop codon recognition. In omnipotent eRF1s, stop codon recognition is associated with the N-terminal domain of the protein. Using both in vitro and in vivo assays, we show here that chimeric molecules composed of the N-terminal domain of Stylonychia eRF1 fused to the core domain (MC domain) of human eRF1 retained specificity toward UGA; this unambiguously associates eRF1 stop codon specificity to the nature of its N-terminal domain. Functional analysis of eRF1 chimeras constructed by swapping ciliate N-terminal domain sequences with the matching ones from the human protein highlighted the crucial role of the tripeptide QFM in restricting Stylonychia eRF1 specificity toward UGA. Using the site-directed mutagenesis, we show that Paramecium eRF1 specificity toward UGA resides within the NIKS (amino acids 61-64) and YxCxxxF (amino acids 124-131) motifs. Thus, we establish that eRF1 from two different ciliates relies on different molecular mechanisms to achieve specificity toward the UGA stop codon. This finding suggests that eRF1 restriction of specificity to only UGA might have been an early event occurring in independent instances in ciliate evolutionary history, possibly facilitating the reassignment of UAG and UAA to sense codons.
Asunto(s)
Cilióforos/genética , Codón de Terminación , Factores de Terminación de Péptidos/genética , Biosíntesis de Proteínas/genética , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Humanos , Paramecium/genética , Factores de Terminación de Péptidos/fisiología , Proteínas Protozoarias/fisiología , Proteínas Recombinantes de Fusión , Especificidad por SustratoRESUMEN
The mechanisms leading to non-lethality of nonsense mutations in essential genes are poorly understood. Here, we focus on the factors influencing viability of yeast cells bearing premature termination codons (PTCs) in the essential gene SUP45 encoding translation termination factor eRF1. Using a dual reporter system we compared readthrough efficiency of the natural termination codon of SUP45 gene, spontaneous sup45-n (nonsense) mutations, nonsense mutations obtained by site-directed mutagenesis (76Q --> TAA, 242R --> TGA, 317L --> TAG). The nonsense mutations in SUP45 gene were shown to be situated in moderate contexts for readthrough efficiency. We showed that readthrough efficiency of some of the mutations present in the sup45 mutants is not correlated with full-length Sup45 protein amount. This resulted from modification of both sup45 mRNA stability which varies 3-fold among sup45-n mutants and degradation rate of mutant Sup45 proteins. Our results demonstrate that some substitutions in the place of PTCs decrease Sup45 stability. The viability of sup45 nonsense mutants is therefore supported by diverse mechanisms that control the final amount of functional Sup45 in cells.
Asunto(s)
Codón sin Sentido , Genes Fúngicos , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , ADN de Hongos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Mutagénesis Sitio-Dirigida , Biosíntesis de Proteínas , Estabilidad del ARN , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genéticaRESUMEN
BACKGROUND: When a stop codon is located in the ribosomal A-site, the termination complex promotes release of the polypeptide and dissociation of the 80S ribosome. In eukaryotes two proteins eRF1 and eRF3 play a crucial function in the termination process. The essential GTPase Sup35p, the eRF3 release factor of Saccharomyces cerevisiae is highly conserved. In particular, we observed that all eRF3 homologs share a potential phosphorylation site at threonine 341, suggesting a functional role for this residue. The goal of this study was to determine whether this residue is actually phosphorylated in yeast and if it is involved in the termination activity of the protein. RESULTS: We detected no phosphorylation of the Sup35 protein in vivo. However, we show that it is phosphorylated by the cAMP-dependent protein kinase A on T341 in vitro. T341 was mutated to either alanine or to aspartic acid to assess the role of this residue in the activity of the protein. Both mutant proteins showed a large decrease of GTPase activity and a reduced interaction with eRF1/Sup45p. This was correlated with an increase of translational readthrough in cells carrying the mutant alleles. We also show that this residue is involved in functional interaction between the N- and C-domains of the protein. CONCLUSION: Our results point to a new critical residue involved in the translation termination activity of Sup35 and in functional interaction between the N- and C-domains of the protein. They also raise interesting questions about the relation between GTPase activity of Sup35 and its essential function in yeast.
Asunto(s)
GTP Fosfohidrolasas/genética , Mutación , Terminación de la Cadena Péptídica Traduccional/genética , Priones/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , GTP Fosfohidrolasas/metabolismo , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Fosforilación , Priones/metabolismo , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
In eukaryotes, release factors 1 and 3 (eRF1 and eRF3) are recruited to promote translation termination when a stop codon on the mRNA enters at the ribosomal A-site. However, their overexpression increases termination efficiency only moderately, suggesting that other factors might be involved in the termination process. To determine such unknown components, we performed a genetic screen in Saccharomyces cerevisiae that identified genes increasing termination efficiency when overexpressed. For this purpose, we constructed a dedicated reporter strain in which a leaky stop codon is inserted into the chromosomal copy of the ade2 gene. Twenty-five antisuppressor candidates were identified and characterized for their impact on readthrough. Among them, SSB1 and snR18, two factors close to the exit tunnel of the ribosome, directed the strongest antisuppression effects when overexpressed, showing that they may be involved in fine-tuning of the translation termination level.
Asunto(s)
Terminación de la Cadena Péptídica Traduccional , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Carboxiliasas/genética , Codón de Terminación , ADN de Hongos/genética , Expresión Génica , Genes Fúngicos , Genes Reporteros , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Sustancias Macromoleculares , Modelos Moleculares , Mutagénesis , Factor 1 de Elongación Peptídica/genética , Factor 1 de Elongación Peptídica/metabolismo , ARN de Hongos/química , ARN de Hongos/genética , ARN Nucleolar Pequeño/química , ARN Nucleolar Pequeño/genética , Ribosomas/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Pyrrolysyl-tRNA synthetase and its cognate suppressor tRNA(Pyl) mediate pyrrolysine (Pyl) insertion at in frame UAG codons. The presence of an RNA hairpin structure named Pyl insertion structure (PYLIS) downstream of the suppression site has been shown to stimulate the insertion of Pyl in archaea. We study here the impact of the presence of PYLIS on the level of Pyl and the Pyl analog N-epsilon-cyclopentyloxycarbonyl-l-lysine (Cyc) incorporation using a quantitative lacZ-luc tandem reporter system in an Escherichia coli context. We show that PYLIS has no effect on the level of neither Pyl nor Cyc incorporation. Exogenously supplying our reporter system with d-ornithine significantly increases suppression efficiency, indicating that d-ornithine is a direct precursor to Pyl.
Asunto(s)
Escherichia coli/genética , Código Genético , Lisina/análogos & derivados , Proteínas Arqueales/genética , Secuencia de Bases , Cartilla de ADN/genética , ADN Bacteriano/genética , Escherichia coli/metabolismo , Genes Arqueales , Genes Bacterianos , Lisina/genética , Lisina/metabolismo , Methanosarcina barkeri/genética , Metiltransferasas/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Ornitina/metabolismo , ARN Bacteriano/química , ARN Bacteriano/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
BACKGROUND: Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which acts as a chloride channel activated by cyclic AMP (cAMP). The most frequent mutation found in 70% of CF patients is F508del, while premature stop mutations are found in about 10% of patients. In vitro aminoglycoside antibiotics (e.g. gentamicin) suppress nonsense mutations located in CFTR permitting translation to continue to the natural termination codon. Pharmacologic suppression of stop mutations within the CFTR may be of benefit to a significant number of patients. Our pilot study was conducted to determine whether intravenous gentamicin suppresses stop codons in CF patients and whether it has clinical benefits. METHODS: A dual gene reporter system was used to determine the gentamicin-induced readthrough level of the most frequent stop mutations within the CFTR in the French population. We investigated readthrough efficiency in response to 10 mg/kg once-daily intravenous gentamicin perfusions in patients with and without stop mutations. Respiratory function, sweat chloride concentration, nasal potential difference (NPD) and CFTR expression in nasal epithelial cells were measured at baseline and after 15 days of treatment. RESULTS: After in vitro gentamicin incubation, the readthrough efficiency for the Y122X mutation was at least five times higher than that for G542X, R1162X, and W1282X. In six of the nine patients with the Y122X mutation, CFTR immunodetection showed protein at the membrane of the nasal epithelial cells and the CFTR-dependent Cl- secretion in NPD measurements increased significantly. Respiratory status also improved in these patients, irrespective of the gentamicin sensitivity of the bacteria present in the sputum. Mean sweat chloride concentration decreased significantly and normalised in two patients. Clinical status, NPD and sweat Cl- values did not change in the Y122X patients with no protein expression, in patients with the other stop mutations investigated in vitro and those without stop mutations. CONCLUSION: Suppression of stop mutations in the CFTR gene with parenteral gentamicin can be predicted in vitro and is associated with clinical benefit and significant modification of the CFTR-mediated Cl- transport in nasal and sweat gland epithelium.
Asunto(s)
Antibacterianos/uso terapéutico , Codón sin Sentido , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/tratamiento farmacológico , Gentamicinas/uso terapéutico , Adolescente , Adulto , Células Cultivadas , Niño , Cloruros/metabolismo , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Genes Reporteros , Gentamicinas/administración & dosificación , Humanos , Inyecciones Intravenosas , Mutación , Proyectos Piloto , Biosíntesis de Proteínas/efectos de los fármacosRESUMEN
The aim of this approach was to identify the major determinants, located at the 5' end of the stop codon, that modulate translational read-through in Saccharomyces cerevisiae. We developed a library of oligonucleotides degenerate at the six positions immediately upstream of the termination codon, cloned in the ADE2 reporter gene. Variations at these positions modulated translational read-through efficiency approximately 16-fold. The major effect was imposed by the two nucleotides immediately upstream of the stop codon. We showed that this effect was neither mediated by the last amino acid residues present in the polypeptide chain nor by the tRNA present in the ribosomal P site. We propose that the mRNA structure, depending on the nucleotides in the P site, is the main 5' determinant of read-through efficiency.
Asunto(s)
Nucleótidos de Adenina/genética , Codón de Terminación/genética , Extensión de la Cadena Peptídica de Translación/genética , Secuencias Reguladoras de Ácido Ribonucleico/genética , Saccharomyces cerevisiae/genética , Composición de Base , Secuencia de Bases , Sitios de Unión , Biblioteca de Genes , Genes Fúngicos/genética , Genes Reporteros/genética , Conformación de Ácido Nucleico , Oligorribonucleótidos/química , Oligorribonucleótidos/genética , Oligorribonucleótidos/metabolismo , ARN de Hongos/química , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
We specifically sought genes within the yeast genome controlled by a non-conventional translation mechanism involving the stop codon. For this reason, we designed a computer program using the yeast database genomic regions, and seeking two adjacent open reading frames separated only by a unique stop codon (called SORFs). Among the 58 SORFs identified, eight displayed a stop codon bypass level ranging from 3 to 25%. For each of the eight sequences, we demonstrated the presence of a poly(A) mRNA. Using isogenic [PSI(+)] and [psi(-)] yeast strains, we showed that for two of the sequences the mechanism used is a bona fide readthrough. However, the six remaining sequences were not sensitive to the PSI state, indicating either a translation termination process independent of eRF3 or a new stop codon bypass mechanism. Our results demonstrate that the presence of a stop codon in a large ORF may not always correspond to a sequencing error, or a pseudogene, but can be a recoding signal in a functional gene. This emphasizes that genome annotation should take into account the fact that recoding signals could be more frequently used than previously expected.
Asunto(s)
Codón de Terminación/genética , Genes Fúngicos/genética , Saccharomyces cerevisiae/genética , Programas Informáticos , Secuencia de Bases , Operón Lac/genética , Luciferasas/genética , Luciferasas/metabolismo , Sistemas de Lectura Abierta/genética , Plásmidos/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Ácido Nucleico , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoAsunto(s)
Aminoglicósidos/farmacología , Apoptosis/genética , Codón sin Sentido/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes p53 , Terapia Genética/métodos , Neoplasias/genética , Estabilidad del ARN/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Aminoglicósidos/uso terapéutico , Regulación Neoplásica de la Expresión Génica/genética , Gentamicinas/farmacología , Humanos , Neoplasias Pulmonares/patología , Neoplasias/terapia , Mutación Puntual , Estabilidad del ARN/genética , Ribosomas/efectos de los fármacos , Transcripción Genética/genética , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
In eukaryotes, translation termination is dependent on the availability of both release factors, eRF1 and eRF3; however, the precise mechanisms involved remain poorly understood. In particular, the fact that the phenotype of release factor mutants is pleiotropic could imply that other factors and interactions are involved in translation termination. To identify unknown elements involved in this process, we performed a genetic screen using a reporter strain in which a leaky stop codon is inserted in the lacZ reporter gene, attempting to isolate factors modifying termination efficiency when overexpressed. Twelve suppressors and 11 antisuppressors, increasing or decreasing termination readthrough, respectively, were identified and analyzed for three secondary phenotypes often associated with translation mutations: thermosensitivity, G418 sensitivity, and sensitivity to osmotic pressure. Interestingly, among these candidates, we identified two genes, SSO1 and STU2, involved in protein transport and spindle pole body formation, respectively, suggesting puzzling connections with the translation termination process.
Asunto(s)
Biosíntesis de Proteínas/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Citoesqueleto/fisiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Expresión Génica , Vectores Genéticos , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/fisiología , Biosíntesis de Proteínas/fisiología , Transporte de Proteínas , Proteínas Qa-SNARE , ARN de Transferencia de Glutamina/genética , ARN de Transferencia de Glutamina/fisiología , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
In the model fungus Podospora anserina, the PaYIP3 gene encoding the orthologue of the Saccharomyces cerevisiae YIP3 Rab-GDI complex dissociation factor expresses two polypeptides, one of which, the long form, is produced through a programmed translation frameshift. Inactivation of PaYIP3 results in slightly delayed growth associated with modification in repartition of fruiting body on the thallus, along with reduced ascospore production on wood. Long and short forms of PaYIP3 are expressed in the mycelium, while only the short form appears expressed in the maturing fruiting body (perithecium). The frameshift has been conserved over the evolution of the Pezizomycotina, lasting for over 400 million years, suggesting that it has an important role in the wild.