RESUMEN
ß-dystroglycan (ß-DG) is a key component of multiprotein complexes in the plasma membrane and nuclear envelope. In addition, ß-DG undergoes two successive proteolytic cleavages that result in the liberation of its intracellular domain (ICD) into the cytosol and nucleus. However, stimuli-inducing ICD cleavage and the physiological relevance of this proteolytic fragment are largely unknown. In this study we show for the first time that ß-DG ICD is targeted to the nucleolus where it interacts with the nuclear proteins B23 and UBF (central factor of Pol I-mediated rRNA gene transcription) and binds to rDNA promoter regions. Interestingly DG silencing results in reduced B23 and UBF levels and aberrant nucleolar morphology. Furthermore, ß-DG ICD cleavage is induced by different nucleolar stressors, including oxidative stress, acidosis, and UV irradiation, which implies its participation in the response to nucleolar stress. Consistent with this idea, overexpression of ß-DG elicited mislocalization and decreased levels of UBF and suppression of rRNA expression, which in turn provoked altered ribosome profiling and decreased cell growth. Collectively our data reveal that ß-DG ICD acts as negative regulator of rDNA transcription by impeding the transcriptional activity of UBF, as a part of the protective mechanism activated in response to nucleolar stress.
Asunto(s)
Nucléolo Celular/metabolismo , Distroglicanos/metabolismo , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , ARN Ribosómico/biosíntesis , Animales , Proliferación Celular/genética , Citoplasma/metabolismo , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Distroglicanos/antagonistas & inhibidores , Distroglicanos/genética , Ratones , Mioblastos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Estrés Oxidativo , Proteínas del Complejo de Iniciación de Transcripción Pol1/genética , Dominios Proteicos/genética , ARN Ribosómico/genética , Ribosomas/metabolismo , Transcripción Genética , Regulación hacia Arriba/genéticaRESUMEN
In Schizosaccharomyces pombe, ribosomal protein gene (RPG) promoters contain a TATA box analog, the HomolD box, which is bound by the Rrn7 protein. Despite the importance of ribosome biogenesis for cell survival, the mechanisms underlying RPG transcription remain unknown. In this study, we found that components of the RNA polymerase II (RNAPII) system, consisting of the initiation or general transcription factors (GTFs) TFIIA, IIB, IIE, TATA-binding protein (TBP) and the RNAPII holoenzyme, interacted directly with Rrn7 in vitro, and were able to form a preinitiation complex (PIC) on the HomolD box. PIC complex formation follows an ordered pathway on these promoters. The GTFs and RNAPII can also be cross-linked to HomolD-containing promoters in vivo. In an in vitro reconstituted transcription system, RNAPII components and Rrn7 were necessary for HomolD-directed transcription. The Mediator complex was required for basal transcription from those promoters in whole cell extract (WCE). The Med17 subunit of Mediator also can be cross-linked to the promoter region of HomolD-containing promoters in vivo, suggesting the presence of the Mediator complex on HomolD box-containing promoters. Together, these data show that components of the RNAPII machinery and Rrn7 participate in the PIC assembly on the HomolD box, thereby directing RPG transcription.
Asunto(s)
Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Proteínas del Complejo de Iniciación de Transcripción Pol1/genética , Proteínas Ribosómicas/genética , Schizosaccharomyces/genética , TATA Box , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/metabolismo , Expresión Génica , Complejo Mediador/genética , Complejo Mediador/metabolismo , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribosómicas/metabolismo , Schizosaccharomyces/metabolismo , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIIA/genética , Factor de Transcripción TFIIA/metabolismo , Factor de Transcripción TFIIB/genética , Factor de Transcripción TFIIB/metabolismo , Factores de Transcripción TFII/genética , Factores de Transcripción TFII/metabolismo , Transcripción GenéticaRESUMEN
We isolated two TATA-binding protein-associated factor (TAF) genes, TAF10 and TAF13, from pepper (Capsicum annuum). The complete coding sequences were amplified using reverse transcriptase-PCR on the basis of conserved sequence information of eggplant and several other plant species. Nucleotide sequence analysis of these two genes revealed that the pepper TAF10 gene encodes a protein of 103 amino acids that belongs to the TAF10 superfamily. The pepper TAF10 gene was highly expressed in the pericarp and placenta, moderately expressed in the stems, flowers, seeds and leaves, and weakly expressed in roots. The TAF13 gene was found to encode a protein of 130 amino acids that belongs to the TAF13 superfamily. The TAF13 gene was highly expressed in the stems, flowers and pericarp, moderately expressed in the leaves, placenta and seeds, and weakly expressed in roots.
Asunto(s)
Capsicum/genética , Regulación de la Expresión Génica de las Plantas , Factores Asociados con la Proteína de Unión a TATA/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Flores/metabolismo , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , Hojas de la Planta/metabolismo , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Raíces de Plantas/metabolismo , Tallos de la Planta/metabolismo , Proteínas del Complejo de Iniciación de Transcripción Pol1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Semillas/metabolismo , Análisis de Secuencia de ADN , Factores Asociados con la Proteína de Unión a TATA/biosíntesis , Factores de Transcripción TFII/genéticaRESUMEN
The region in promoters that specifies the transcription machinery is called the core promoter, displaying core promoter elements (CPE) necessary for establishment of a preinitiation complex and the initiation of transcription. A classical CPE is the TATA box. In fission yeast, Schizosaccharomyces pombe, a new CPE, called HomolD box, was discovered. Collectively, 141 ribosomal protein genes encoding the full set of 79 different ribosomal proteins and more than 60 other housekeeping genes display a HomolD box in the core promoter. Here, we show that transcription directed by the HomolD box requires the RNA polymerase II machinery, including the general transcription factors. Most intriguingly, however, we identify, by DNA affinity purification, Rrn7 as the protein binding to the HomolD box. Rrn7 is an evolutionary conserved member of the RNA polymerase I machinery involved in transcription initiation of core ribosomal DNA promoters. ChIP shows that Rrn7 cross-links to a ribosomal protein gene promoter containing the HomolD box but not to a promoter containing a TATA box. Taken together, our results suggest that Rrn7 is an excellent candidate to be involved in the coordination of ribosomal DNA and ribosomal gene transcription during ribosome synthesis and, therefore, offer a new perspective to study conservation and evolvability of regulatory networks in eukaryotes.