RESUMEN
Ribosome biogenesis is a major metabolic effort for growing cells. In Saccharomyces cerevisiae, Hmo1, an abundant high-mobility group box protein (HMGB) binds to the coding region of the RNA polymerase I transcribed ribosomal RNAs genes and the promoters of â¼70% of ribosomal protein genes. In this study, we have demonstrated the functional conservation of eukaryotic HMGB proteins involved in ribosomal DNA (rDNA) transcription. We have shown that when expressed in budding yeast, human UBF1 and a newly identified Sp-Hmo1 (Schizosaccharomyces pombe) localize to the nucleolus and suppress growth defect of the RNA polymerase I mutant rpa49-Δ. Owing to the multiple functions of both proteins, Hmo1 and UBF1 are not fully interchangeable. By deletion and domains swapping in Hmo1, we identified essential domains that stimulate rDNA transcription but are not fully required for stimulation of ribosomal protein genes expression. Hmo1 is organized in four functional domains: a dimerization module, a canonical HMGB motif followed by a conserved domain and a C-terminal nucleolar localization signal. We propose that Hmo1 has acquired species-specific functions and shares with UBF1 and Sp-Hmo1 an ancestral function to stimulate rDNA transcription.
Asunto(s)
ADN Ribosómico/metabolismo , Proteínas HMGB/química , Proteínas HMGB/metabolismo , Proteínas del Grupo de Alta Movilidad/química , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Nucléolo Celular/metabolismo , Secuencia Conservada , Proteínas HMGB/genética , Proteínas del Grupo de Alta Movilidad/genética , Humanos , Proteínas del Complejo de Iniciación de Transcripción Pol1/química , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , Estructura Terciaria de Proteína , ARN Polimerasa I/metabolismo , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Relación Estructura-ActividadRESUMEN
The nonrandom positioning of genes inside eukaryotic cell nuclei is implicated in central nuclear functions. However, the spatial organization of the genome remains largely uncharted, owing to limited resolution of optical microscopy, paucity of nuclear landmarks and moderate cell sampling. We developed a computational imaging approach that creates high-resolution probabilistic maps of subnuclear domains occupied by individual loci in budding yeast through automated analysis of thousands of living cells. After validation, we applied the technique to genes involved in galactose metabolism and ribosome biogenesis. We found that genomic loci are confined to 'gene territories' much smaller than the nucleus, which can be remodeled during transcriptional activation, and that the nucleolus is an important landmark for gene positioning. The technique can be used to visualize and quantify territory positions relative to each other and to nuclear landmarks, and should advance studies of nuclear architecture and function.
Asunto(s)
Núcleo Celular/fisiología , Núcleo Celular/ultraestructura , Cromosomas/genética , Cromosomas/ultraestructura , Interpretación Estadística de Datos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Compartimento Celular , Sensibilidad y EspecificidadRESUMEN
The bimolecular fluorescence complementation (BiFC) assay, which allows the investigation of interacting molecules in vivo, was applied to study complex formation between the splicing factor Y14 and nuclear export factor 1 (NXF1), which evidence indicates are functionally associated with nuclear mRNA. Y14 linked to the COOH terminus of yellow fluorescent protein (YFP; YC-Y14), and NXF1 fused to the NH2 terminus of YFP (YN-NXF1) expressed in MCF7 cells yielded BiFC upon specific binding. Fluorescence accumulated within and around nuclear speckles, suggesting the involvement of speckles in mRNA processing and export. Accordingly, BiFC depended on transcription and full-length NXF1. Coimmunoprecipitation of YC-Y14 with YN-NXF1, NXF1, Y14, and RNA indicated that YC-Y14 and YN-NXF1 functionally associate with RNA. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching revealed that roughly half of the accumulated BiFC complexes were immobile in vivo. This immobile fraction was readily depleted by adenosine triphosphate (ATP) administration in permeabilized cells. These results suggest that a fraction of RNA, which remains in the nucleus for several hours despite its association with splicing and export proteins, accumulates in speckles because of an ATP-dependent mechanism.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Mapeo de Interacción de Proteínas , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Digitonina/farmacología , Recuperación de Fluorescencia tras Fotoblanqueo , Humanos , Espacio Intranuclear/metabolismo , Cinética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Mutación/genética , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Unión Proteica , ARN Polimerasa II/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas/metabolismo , Factores de Empalme Serina-Arginina , Azida Sódica/farmacología , TransfecciónRESUMEN
OBJECTIVE: Elevated plasma levels of lipoprotein(a) [Lp(a)], referred to as lipoprotein(a)-hyperlipoproteinemia [Lp(a)-HLP], are an independent risk factor for atherosclerosis. Lipoprotein apheresis (LA) enables an effective reduction of Lp(a) plasma levels. The present study investigates the effects of LA in patients with Lp(a)-HLP and peripheral artery disease (PAD). METHODS: Ten patients with isolated Lp(a)-HLP and severe PAD and who had recently undergone a revascularization (index procedure) were prospectively included in this observational single center study. All patients received weekly LA. Ankle-brachial-index (ABI), transcutaneous partial oxygen pressure (tcpO2), pain level, and walking distance were assessed at baseline and at the follow ups scheduled 1, 3, 6, 12, and 24 months after initiation of LA. The number of revascularizations within 12 months prior and within 24 months after the index procedure was determined. RESULTS: As early as 1 month after initiation of LA, all investigated parameters had improved significantly compared to baseline. This improvement was further substantiated under LA throughout the entire follow-up period. Comparing baseline results with the 24-month follow-up, the average ABI increased from 0.53 ± 0.15 to 0.97 ± 0.08 (P < 0.001). The mean tcpO2 also increased from 42.9 ± 2.3 mmHg to 61 ± 4.6 mmHg (P < 0.001). The improved perfusion led to a reduction of the mean pain level from 7.0 ± 1.5 to 1.1 ± 0.4 (P < 0.001) on a visual analogue scale (VAS) and an extension of the mean walking distance from 87 ± 60 m to 402 ± 119 m (P < 0.001). All patients suffered from severe PAD with a high number of revascularizations in the 12 months prior to the index procedure (35 procedures in 120 patient-months). Since initiation of LA, the number of revascularizations dropped significantly and remained very low during the entire follow-up period (2 procedures in 229 patient-months, P < 0.001). CONCLUSION: In patients with Lp(a)-HLP and severe PAD, LA results in sustained improvement of circulation, pain level and walking distance. The number of repeat revascularizations is strongly reduced under LA treatment.
Asunto(s)
Eliminación de Componentes Sanguíneos/métodos , Hiperlipoproteinemias/terapia , Lipoproteína(a)/sangre , Enfermedad Arterial Periférica/terapia , Índice Tobillo Braquial , Biomarcadores/sangre , Monitoreo de Gas Sanguíneo Transcutáneo , Tolerancia al Ejercicio , Femenino , Estudios de Seguimiento , Humanos , Hiperlipoproteinemias/sangre , Hiperlipoproteinemias/complicaciones , Hiperlipoproteinemias/fisiopatología , Masculino , Persona de Mediana Edad , Dimensión del Dolor , Enfermedad Arterial Periférica/sangre , Enfermedad Arterial Periférica/etiología , Enfermedad Arterial Periférica/fisiopatología , Proyectos Piloto , Estudios Prospectivos , Recuperación de la Función , Flujo Sanguíneo Regional , Factores de Riesgo , Factores de Tiempo , Resultado del Tratamiento , CaminataRESUMEN
OBJECTIVE: Hyperlipoproteinemia(a) [Lp(a)-HLP] is a major risk factor for severe atherosclerosis. The present investigation sought to assess the effect of lipoprotein apheresis (LA) in patients with peripheral artery disease (PAD) and Lp(a)-HLP. METHODS: In January 2013, we started a registry for Lp(a)-HLP patients who receive weekly LA in our center. So far, ten patients with severe PAD and isolated Lp(a)-HLP who recently underwent revascularization (index procedure) have been included. Pain level, ankle-brachial-index (ABI), transcutaneous oxygen pressure (tcpO2) and walking distance were determined before, as well as 1, 3, 6 and 12 months after initiation of LA. Furthermore, the mean time interval between revascularizations within the 12 months prior to the index procedure and up to 12 months after the index procedure was assessed. RESULTS: All analyzed parameters significantly improved under LA. When comparing the results before LA with the results after 12 months, the ankle-brachial-index increased from 0.5 ± 0.2 to 0.9 ± 0.1 (P < 0.001). The tcpO2 levels also increased from 42.9 ± 2.3 mmHg to 59.0 ± 8.9 mmHg (P < 0.001). The improved microcirculation was associated with a reduction of the mean pain level from 7.0 ± 1.5 to 2.0 ± 0.8 (P < 0.001) as determined using the visual analog scale. The walking distance increased from 87 ± 60 m to 313 ± 145 m (P < 0.001). Importantly, the frequency of revascularization procedures was strongly decreased under LA. All patients combined underwent 35 revascularizations within the 12 months prior to the index procedure (mean interval between two revascularizations: 104.3 days). Since the index procedure, only one revascularization was necessary within 79 patient-months under LA (mean interval: 2404.5 days, P < 0.001). CONCLUSION: LA improves circulation, oxygen supply, level of pain and walking distance in patients with severe PAD and Lp(a)-HLP. The frequency of revascularization procedures is strongly reduced under LA treatment.
Asunto(s)
Eliminación de Componentes Sanguíneos/métodos , Hiperlipoproteinemias/terapia , Lipoproteína(a)/sangre , Enfermedad Arterial Periférica/cirugía , Índice Tobillo Braquial , Biomarcadores/sangre , Monitoreo de Gas Sanguíneo Transcutáneo , Prueba de Esfuerzo , Tolerancia al Ejercicio , Femenino , Alemania , Hemodinámica , Humanos , Hiperlipoproteinemias/sangre , Hiperlipoproteinemias/complicaciones , Hiperlipoproteinemias/diagnóstico , Masculino , Microcirculación , Persona de Mediana Edad , Dimensión del Dolor , Enfermedad Arterial Periférica/complicaciones , Enfermedad Arterial Periférica/diagnóstico , Enfermedad Arterial Periférica/fisiopatología , Valor Predictivo de las Pruebas , Estudios Prospectivos , Recuperación de la Función , Sistema de Registros , Factores de Riesgo , Índice de Severidad de la Enfermedad , Factores de Tiempo , Resultado del TratamientoRESUMEN
Ribosome biogenesis requires equimolar amounts of four rRNAs and all 79 ribosomal proteins (RP). Coordinated regulation of rRNA and RP synthesis by eukaryotic RNA polymerases (Pol) I, III, and II is a key requirement for growth control. Using a novel global genetic approach, we showed that the absence of Hmo1 becomes lethal when combined with mutations of components of either the RNA Pol II or Pol I transcription machineries, of specific RP, or of the TOR pathway. Hmo1 directly interacts with both the region transcribed by Pol I and a subset of RP gene promoters. Down-regulation of Hmo1 expression affects RP gene expression. Upon TORC1 inhibition, Hmo1 dissociates from ribosomal DNA (rDNA) and some RP gene promoters simultaneously. Finally, in the absence of Hmo1, TOR-dependent repression of RP genes is alleviated. Therefore, we show here that Saccharomyces cerevisiae Hmo1 is directly involved in coordinating rDNA transcription by Pol I and RP gene expression by Pol II under the control of the TOR pathway.