RESUMEN
Saccharomyces cerevisiae has been used as a model system to investigate the mechanisms of pre-mRNA splicing but only a few examples of alternative splice site usage have been described in this organism. Using RNA-Seq analysis of nonsense-mediated mRNA decay (NMD) mutant strains, we show that many S. cerevisiae intron-containing genes exhibit usage of alternative splice sites, but many transcripts generated by splicing at these sites are non-functional because they introduce premature termination codons, leading to degradation by NMD. Analysis of splicing mutants combined with NMD inactivation revealed the role of specific splicing factors in governing the use of these alternative splice sites and identified novel functions for Prp17p in enhancing the use of branchpoint-proximal upstream 3' splice sites and for Prp18p in suppressing the usage of a non-canonical AUG 3'-splice site in GCR1. The use of non-productive alternative splice sites can be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during stress. These results show that alternative splicing is frequent in S. cerevisiae but masked by RNA degradation and that the use of alternative splice sites in this organism is mostly aimed at controlling transcript levels rather than increasing proteome diversity.
Asunto(s)
Empalme Alternativo/genética , Degradación de ARNm Mediada por Codón sin Sentido/genética , Sitios de Empalme de ARN/genética , Empalme del ARN/genética , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Codón sin Sentido/genética , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/genética , Regiones Promotoras Genéticas/genética , ARN/genética , Factores de Empalme de ARN , Estabilidad del ARN/genética , Proteínas de Unión al ARN/genética , Ribonucleoproteína Nuclear Pequeña U5/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
Coenzyme Qn (ubiquinone or Qn) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail of n isoprene units. Saccharomyces cerevisiae coq1-coq9 mutants have defects in Q biosynthesis, lack Q6, are respiratory defective, and sensitive to stress imposed by polyunsaturated fatty acids. The hallmark phenotype of the Q-less yeast coq mutants is that respiration in isolated mitochondria can be rescued by the addition of Q2, a soluble Q analog. Yeast coq10 mutants share each of these phenotypes, with the surprising exception that they continue to produce Q6. Structure determination of the Caulobacter crescentus Coq10 homolog (CC1736) revealed a steroidogenic acute regulatory protein-related lipid transfer (START) domain, a hydrophobic tunnel known to bind specific lipids in other START domain family members. Here we show that purified CC1736 binds Q2, Q3, Q10, or demethoxy-Q3 in an equimolar ratio, but fails to bind 3-farnesyl-4-hydroxybenzoic acid, a farnesylated analog of an early Q-intermediate. Over-expression of C. crescentus CC1736 or COQ8 restores respiratory electron transport and antioxidant function of Q6 in the yeast coq10 null mutant. Studies with stable isotope ring precursors of Q reveal that early Q-biosynthetic intermediates accumulate in the coq10 mutant and de novo Q-biosynthesis is less efficient than in the wild-type yeast or rescued coq10 mutant. The results suggest that the Coq10 polypeptide:Q (protein:ligand) complex may serve essential functions in facilitating de novo Q biosynthesis and in delivering newly synthesized Q to one or more complexes of the respiratory electron transport chain.
Asunto(s)
Antioxidantes/metabolismo , Transporte de Electrón/fisiología , Péptidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquinona/metabolismo , Secuencia de Aminoácidos , Transporte de Electrón/genética , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Ubiquinona/análogos & derivadosRESUMEN
The MATa1 gene encodes a transcriptional repressor that is an important modulator of sex-specific gene expression in Saccharomyces cerevisiae. MATa1 contains two small introns, both of which need to be accurately excised for proper expression of a functional MATa1 product and to avoid production of aberrant forms of the repressor. Here, we show that unspliced and partially spliced forms of the MATa1 mRNA are degraded by the nuclear exonuclease Rat1p, the nuclear exosome and by the nuclear RNase III endonuclease Rnt1p to prevent undesired expression of non-functional a1 proteins. In addition, we show that mis-spliced forms of MATa1 in which the splicing machinery has skipped exon2 and generated exon1-exon3 products are degraded by the nuclear 5'-3' exonuclease Rat1p and by the nuclear exosome. This function for Rat1p and the nuclear exosome in the degradation of exon-skipped products is also observed for three other genes that contain two introns (DYN2, SUS1, YOS1), identifying a novel nuclear quality control pathway for aberrantly spliced RNAs that have skipped exons.
Asunto(s)
Núcleo Celular/enzimología , Exones , Proteínas de Homeodominio/genética , Empalme del ARN , Estabilidad del ARN , Proteínas Represoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Exorribonucleasas/genética , Proteínas de Homeodominio/metabolismo , Intrones , Mutación , Sitios de Empalme de ARN , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Ribonucleasa III/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
INTRODUCTION: The aim of the present study was to investigate whether the concentration of arsenic (As) released from gray or white mineral trioxide aggregates (MTAs) met the requirement of the International Standards Organization (ISO) for dental cements. METHODS: Sample preparations were carried out according to the ISO methods. After centrifugation of dissolved samples, As (III) concentration in the final supernatant was analyzed by a high-performance atomic absorption spectrophotometer. RESULTS: As (III) concentration from both MTAs was much less than the required value (2 ppm) for dental cements regulated by the ISO. An experiment simulating pulp capping by using MTA revealed that As concentration was also below the standard value of the ISO. The As concentration in white MTA was lower than the value (10 ppb) recommended for tap water and environmental standards. CONCLUSIONS: The present in vitro studies demonstrated that there is no threat to patient health in using commercially available brands of MTA for endodontic practices.
Asunto(s)
Compuestos de Aluminio/química , Arsénico/análisis , Compuestos de Calcio/química , Óxidos/química , Materiales de Obturación del Conducto Radicular/química , Silicatos/química , Cementos Dentales/normas , Combinación de Medicamentos , Estándares de Referencia , Espectrofotometría AtómicaRESUMEN
The role of many splicing factors in pre-mRNA splicing and the involvement of these factors in the processing of specific transcripts have often been defined through the analysis of loss-of-function mutants in vivo. Here we show that inactivating the nonsense-mediated mRNA decay (NMD) results in an enhancement of splicing phenotypes associated with several S. cerevisiae splicing factor mutations. Tiling microarrays showed that inactivation of the NMD factor Upf1p in the prp17Delta and prp18Delta mutant strains results in a larger spectrum of splicing defects than what is observed in the single mutants, including new transcripts previously shown unaffected by Prp17p or Prp18p inactivation. Inactivation of Upf1p in the second step/recycling factor prp22-1 mutant and in the nam8Delta and mud1Delta U1 snRNP component mutants also increase unspliced precursor accumulation of several specific transcripts. In addition, deletion of UPF1 partially suppresses the growth defects associated with the prp17Delta or prp22-1 mutations, demonstrating a positive genetic interaction between NMD and splicing factor mutants. These results show that RNA surveillance by NMD can mask some of the effects of splicing factor mutations, and that the roles of splicing factors cannot be fully understood in vivo unless RNA degradation systems that degrade unspliced precursors are also inactivated.
Asunto(s)
Codón sin Sentido/genética , Mutación , Empalme del ARN , Estabilidad del ARN , Empalmosomas/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Codón sin Sentido/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Factores de Empalme de ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U5/genética , Ribonucleoproteína Nuclear Pequeña U5/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Empalmosomas/metabolismo , Transcripción GenéticaRESUMEN
We encountered an unusual case of hyperparathyroidism with both hemosiderin deposits on the ribs and low intensity on T2-weighted magnetic resonance imaging (MRI) caused by a parathyroid adenoma with multiple brown tumors that mimicked metastatic bone tumor due to false positive results on computed tomography (CT) and Tc-99m sestamibi (MIBI) imaging. The patient, a middle-aged woman, had very high serum levels of calcium (14.1 mg/dl), alkaline phosphatase (9,369 IU/l) and intact-PTH (12,400 pg/ml), and a large tumor (2.5 cm in diameter) in the lower portion of the left lobe of the thyroid. Plain X-ray revealed a soft tumor in the left chest wall. On CT scan, there were multiple destructive masses in the ribs, including large intramedullary masses on both 3rd ribs. On MIBI scintigraphy, there was strong late uptake in the lower portion of the left cervical region, both 3rd ribs, and the left 7th, 8th, and 10th ribs. T2-weighted image MRI scans showed that both 3rd ribs had a low intensity with hemosiderin deposits. These findings suggested that the patient had hyperparathyroidism with multiple bone metastases due to carcinoma of the parathyroid gland. However, on pathology, the resected tumor of lower portion of the left lobe of thyroid was diagnosed as a parathyroid adenoma, and the tumors of the left 3rd and 7th ribs, as well as the right 2nd rib, were shown to be brown tumors. After resection, the patient's serum levels of calcium, alkaline phosphatase, and intact-PTH normalized. At 1.5-years follow-up, CT, MIBI, and MRI scans showed no abnormal findings. It is necessary to determine whether MRI can be used to distinguish between brown tumors and metastases caused by carcinoma of the parathyroid gland.