RESUMEN
The maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , ADN/metabolismo , Hibridación de Ácido Nucleico , Edición de ARN , ARN/metabolismo , Adenosina Desaminasa/genética , Proteína BRCA1/metabolismo , Línea Celular Tumoral , ADN Helicasas/metabolismo , Eliminación de Gen , Genes Reporteros , Inestabilidad Genómica , Proteínas Fluorescentes Verdes/metabolismo , Recombinación Homóloga/genética , Humanos , Enzimas Multifuncionales/metabolismo , Estabilidad Proteica , ARN Helicasas/metabolismo , Proteínas de Unión al ARN/genética , Proteína de Replicación A/metabolismoRESUMEN
Cytoskeletal proteins are beginning to be considered as key regulators of nuclear function. Among them, actin and myosin have been implicated in numerous tasks, including chromatin regulation, transcription and assembly of nascent ribonucleoprotein complexes. We also know from work performed by several labs that influx of actin and myosin into the nucleus and out of the nucleus is tightly regulated. In particular, in the case of actin, its nucleocytoplasmic import/export cycle is controlled by the importin/exportin system and it correlates with the transcriptional state of the cell. These basic molecular functions of both actin and myosin seem to impact key cellular functions, including development and differentiation as well as the cellular response to DNA damage by directly affecting transcriptional reprograming. These observations are beginning to suggest that actin and myosin could play an important role in consolidating the organization of the mammalian genome and that loss of actin and myosin likely leads to a general instability of the genome. In this chapter, we provide a general background on evidence that actin and myosin are important in key nuclear functions. Following this, we will focus on evidence supporting of a role in genome organization and finally we will discuss increasingly striking results on the role of actin and myosin in the maintenance of genome integrity.
Asunto(s)
Actinas/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Inestabilidad Genómica , Miosinas/metabolismo , Animales , HumanosRESUMEN
Nuclear myosin 1 (NM1) has been implicated in key nuclear functions. Together with actin, it has been shown to initiate and regulate transcription, it is part of the chromatin remodeling complex B-WICH, and is responsible for rearrangements of chromosomal territories in response to external stimuli. Here we show that deletion of NM1 in mouse embryonic fibroblasts leads to chromatin and transcription dysregulation affecting the expression of DNA damage and cell cycle genes. NM1 KO cells exhibit increased DNA damage and changes in cell cycle progression, proliferation, and apoptosis, compatible with a phenotype resulting from impaired p53 signaling. We show that upon DNA damage, NM1 forms a complex with p53 and activates the expression of checkpoint regulator p21 (Cdkn1A) by PCAF and Set1 recruitment to its promoter for histone H3 acetylation and methylation. We propose a role for NM1 in the transcriptional response to DNA damage response and maintenance of genome stability.
Asunto(s)
Núcleo Celular/metabolismo , Ensamble y Desensamble de Cromatina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN , Miosina Tipo I/metabolismo , Transcripción Genética , Animales , Apoptosis , Ciclo Celular , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Núcleo Celular/patología , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Epigénesis Genética , Etopósido/toxicidad , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Ratones , Miosina Tipo I/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
The exosome is a ribonucleolytic complex that plays important roles in RNA metabolism. Here we show that the exosome is necessary for the repair of DNA double-strand breaks (DSBs) in human cells and that RNA clearance is an essential step in homologous recombination. Transcription of DSB-flanking sequences results in the production of damage-induced long non-coding RNAs (dilncRNAs) that engage in DNA-RNA hybrid formation. Depletion of EXOSC10, an exosome catalytic subunit, leads to increased dilncRNA and DNA-RNA hybrid levels. Moreover, the targeting of the ssDNA-binding protein RPA to sites of DNA damage is impaired whereas DNA end resection is hyper-stimulated in EXOSC10-depleted cells. The DNA end resection deregulation is abolished by transcription inhibitors, and RNase H1 overexpression restores the RPA recruitment defect caused by EXOSC10 depletion, which suggests that RNA clearance of newly synthesized dilncRNAs is required for RPA recruitment, controlled DNA end resection and assembly of the homologous recombination machinery.