RESUMEN
Retarded growth and neurodegeneration are hallmarks of the premature aging disease Cockayne syndrome (CS). Cockayne syndrome proteins take part in the key step of ribosomal biogenesis, transcription of RNA polymerase I. Here, we identify a mechanism originating from a disturbed RNA polymerase I transcription that impacts translational fidelity of the ribosomes and consequently produces misfolded proteins. In cells from CS patients, the misfolded proteins are oxidized by the elevated reactive oxygen species (ROS) and provoke an unfolded protein response that represses RNA polymerase I transcription. This pathomechanism can be disrupted by the addition of pharmacological chaperones, suggesting a treatment strategy for CS. Additionally, this loss of proteostasis was not observed in mouse models of CS.
Asunto(s)
Síndrome de Cockayne/patología , Proteostasis , Animales , Línea Celular , Síndrome de Cockayne/genética , Estrés del Retículo Endoplásmico , Humanos , Ratones , Mutación/genética , Estrés Oxidativo , Biosíntesis de Proteínas , Pliegue de Proteína , ARN Polimerasa I/genética , Especies Reactivas de Oxígeno/metabolismo , Transcripción Genética , Xerodermia Pigmentosa/genética , Xerodermia Pigmentosa/patologíaRESUMEN
The nucleolus has long been considered to be a pure ribosome factory. However, over the last two decades it became clear that the nucleolus is involved in numerous other functions besides ribosome biogenesis. Our experiments indicate that the activity of RNA polymerase I (Pol I) transcription monitors the integrity of the DNA and influences the response to nucleolar stress as well as the rate of survival. Cells with a repressed ribosomal DNA (rDNA) transcription activity showed an increased and prolonged p53 stabilisation after UVC-irradiation. Furthermore, p53 stabilisation after inhibition and especially after UVC-irradiation might be due to abrogation of the HDM2-p53 degradation pathway by ribosomal proteins (RPs). Apoptosis mediated by highly activated p53 is a typical hallmark of Cockayne syndrome cells and transcriptional abnormalities and the following activation of the RP-HDM2-p53 pathway would be a possible explanation.
Asunto(s)
ARN Polimerasa I/metabolismo , Transcripción Genética/efectos de la radiación , Rayos Ultravioleta , Apoptosis/efectos de la radiación , Línea Celular , Células HCT116 , Humanos , Proteínas del Complejo de Iniciación de Transcripción Pol1/antagonistas & inhibidores , Proteínas del Complejo de Iniciación de Transcripción Pol1/genética , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , Estabilidad Proteica/efectos de la radiación , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Interferencia de ARN , ARN Polimerasa I/genética , ARN Ribosómico/metabolismo , ARN Interferente Pequeño/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
In addition to performing its canonical function, Telomerase Reverse Transcriptase (TERT) has been shown to participate in cellular processes independent of telomerase activity. Furthermore, although TERT mainly localizes to Cajal bodies, it is also present within the nucleolus. Because the nucleolus is the site of rDNA transcription, we investigated the possible role of telomerase in regulating RNA polymerase I (Pol I). Here we show that TERT binds to rDNA and stimulates transcription by Pol I during liver regeneration and Ras-induced hyperproliferation. Moreover, the inhibition of telomerase activity by TERT- or TERC-specific RNA interference, the overexpression of dominant-negative-TERT, and the application of the telomerase inhibitor imetelstat reduce Pol I transcription and the growth of tumour cells. In vitro, telomerase can stimulate the formation of the transcription initiation complex. Our results demonstrate how non-canonical features of telomerase may direct Pol I transcription in oncogenic and regenerative hyperproliferation.
Asunto(s)
Proliferación Celular/fisiología , ADN Ribosómico/genética , ADN Ribosómico/fisiología , ARN Polimerasa I/fisiología , Telomerasa/fisiología , Transcripción Genética/fisiología , Animales , Línea Celular , Línea Celular Tumoral , Proliferación Celular/genética , Neoplasias Colorrectales/patología , Humanos , Riñón/citología , Hígado/citología , Regeneración Hepática/genética , Regeneración Hepática/fisiología , Pulmón/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Miofibroblastos/citología , Unión Proteica/fisiología , ARN Polimerasa I/genética , Conejos , Telomerasa/genética , Transcripción Genética/genéticaRESUMEN
Mutations in the Cockayne syndrome A (CSA) protein account for 20% of Cockayne syndrome (CS) cases, a childhood disorder of premature aging and early death. Hitherto, CSA has exclusively been described as DNA repair factor of the transcription-coupled branch of nucleotide excision repair. Here we show a novel function of CSA as transcription factor of RNA polymerase I in the nucleolus. Knockdown of CSA reduces pre-rRNA synthesis by RNA polymerase I. CSA associates with RNA polymerase I and the active fraction of the rDNA and stimulates re-initiation of rDNA transcription by recruiting the Cockayne syndrome proteins TFIIH and CSB. Moreover, compared with CSA deficient parental CS cells, CSA transfected CS cells reveal significantly more rRNA with induced growth and enhanced global translation. A previously unknown global dysregulation of ribosomal biogenesis most likely contributes to the reduced growth and premature aging of CS patients.
Asunto(s)
Enzimas Reparadoras del ADN/metabolismo , ARN Polimerasa I/metabolismo , Ribosomas/metabolismo , Factores de Transcripción/metabolismo , Línea Celular Tumoral , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/genética , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de Unión a Poli-ADP-Ribosa , ARN Polimerasa I/genética , Precursores del ARN/biosíntesis , Precursores del ARN/metabolismo , ARN Ribosómico/biosíntesis , ARN Ribosómico/metabolismo , Factor de Transcripción TFIIH/metabolismo , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
TFIIH is a multisubunit factor essential for transcription initiation and promoter escape of RNA polymerase II and for the opening of damaged DNA double strands in nucleotide excision repair (NER). In this study, we have analyzed at which step of the transcription cycle TFIIH is essential for transcription by RNA polymerase I. We demonstrate that TFIIH associates with the rDNA promoter and gene-internal sequences and leaves the rDNA promoter in a complex with RNA polymerase I after start of transcription. Moreover, mutations in the TFIIH subunits XPB and XPD found in Cockayne syndrome impair the interaction of TFIIH with the rDNA, but do not influence initiation complex formation or promoter escape of RNA polymerase I, but preclude the productivity of the enzyme by reducing transcription elongation in vivo and in vitro. Our results implicate that reduced RNA polymerase I transcription elongation and ribosomal stress could be one factor contributing to the Cockayne syndrome phenotype.
