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
RNA polymerase II is recruited to DNA double-strand breaks (DSBs), transcribes the sequences that flank the break and produces a novel RNA type that has been termed damage-induced long non-coding RNA (dilncRNA). DilncRNAs can be processed into short, miRNA-like molecules or degraded by different ribonucleases. They can also form double-stranded RNAs or DNA:RNA hybrids. The DNA:RNA hybrids formed at DSBs contribute to the recruitment of repair factors during the early steps of homologous recombination (HR) and, in this way, contribute to the accuracy of the DNA repair. However, if not resolved, the DNA:RNA hybrids are highly mutagenic and prevent the recruitment of later HR factors. Here recent discoveries about the synthesis, processing, and degradation of dilncRNAs are revised. The focus is on RNA clearance, a necessary step for the successful repair of DSBs and the aim is to reconcile contradictory findings on the effects of dilncRNAs and DNA:RNA hybrids in HR.
Asunto(s)
Roturas del ADN de Doble Cadena , ARN , ADN/genética , Reparación del ADN , Recombinación Homóloga , ARN/genéticaRESUMEN
Best disease, also known as Best vitelliform macular dystrophy, is an autosomal dominant form of macular degeneration. Here, we have generated an induced pluripotent stem cell (iPSC) line derived from a Best disease patient carrying a new dominant mutation in the BEST1 gene. Skin fibroblasts were reprogrammed to iPSCs by the non-integrative Sendai-virus method. The iPSC line has been characterized preserving the BEST1 mutation, expressing the pluripotency markers and being capable to differentiate to endoderm, mesoderm and ectoderm in vitro.
Asunto(s)
Bestrofinas/genética , Línea Celular/citología , Células Madre Pluripotentes Inducidas/metabolismo , Distrofia Macular Viteliforme/genética , Adulto , Bestrofinas/metabolismo , Diferenciación Celular , Línea Celular/metabolismo , Células Cultivadas , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Mutación , Distrofia Macular Viteliforme/metabolismo , Distrofia Macular Viteliforme/fisiopatologíaRESUMEN
Incomplete achromatopsia (ACHM) is a disorder in which there is function defect of cone photoreceptors in the retina and individuals with such disease retain residual color vision. Here, we have generated an induced pluripotent stem cell (iPSC) line carrying a homozygous mutation in the PDE6C gene, already related with this vision disorder. Skin fibroblasts from a patient with incomplete ACHM were reprogrammed to iPSCs by the non-integrative Sendai-virus method. Finally, the iPSC line has been characterized expressing the pluripotency markers and being capable to differentiate to endoderm, mesoderm and ectoderm in vitro.
Asunto(s)
Línea Celular/citología , Defectos de la Visión Cromática/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Proteínas del Ojo/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Células Madre Pluripotentes Inducidas/metabolismo , Adulto , Diferenciación Celular , Línea Celular/metabolismo , Células Cultivadas , Defectos de la Visión Cromática/metabolismo , Defectos de la Visión Cromática/fisiopatología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Proteínas del Ojo/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/fisiopatología , Homocigoto , Humanos , Células Madre Pluripotentes Inducidas/citología , Masculino , MutaciónRESUMEN
Retinitis pigmentosa (RP) is an inherited retinal dystrophy characterized by the progressive degeneration of photoreceptors. In the present study, we have generated an induced pluripotent stem cell (iPSC) line derived from a RP patient with a dominant mutation in the RHO gene, responsible for the synthesis of rhodopsin. The reprogramming of these iPSCs was performed from skin fibroblasts by the Sendai-virus based approach. Characterization of the iPSC line showed a normal karyotype carrying the RHO mutation, expressed pluripotency markers and could be differentiated to endoderm, mesoderm and ectoderm in vitro.
Asunto(s)
Genes Dominantes , Células Madre Pluripotentes Inducidas , Mutación Puntual , Retinitis Pigmentosa , Rodopsinas Sensoriales , Adulto , Línea Celular , Técnicas de Reprogramación Celular , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/metabolismo , Retinitis Pigmentosa/patología , Rodopsinas Sensoriales/genética , Rodopsinas Sensoriales/metabolismo , Piel/metabolismo , Piel/patologíaRESUMEN
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.
Asunto(s)
Roturas del ADN de Doble Cadena , Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Recombinación Homóloga , Proteína de Replicación A/metabolismo , ADN/genética , Exorribonucleasas/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Exosomas/metabolismo , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , ARN Largo no Codificante/genética , ARN Interferente Pequeño/metabolismo , Recombinasa Rad51/metabolismo , Ribonucleasa H/metabolismoRESUMEN
Recent studies suggest that transcription takes place at DNA double-strand breaks (DSBs), that transcripts at DSBs are processed by Drosha and Dicer into damage-induced small RNAs (diRNAs), and that diRNAs are required for DNA repair. However, diRNAs have been mostly detected in reporter constructs or repetitive sequences, and their existence at endogenous loci has been questioned by recent reports. Using the homing endonuclease I-PpoI, we have investigated diRNA production in genetically unperturbed human and mouse cells. I-PpoI is an ideal tool to clarify the requirements for diRNA production because it induces DSBs in different types of loci: the repetitive 28S locus, unique genes and intergenic loci. We show by extensive sequencing that the rDNA locus produces substantial levels of diRNAs, whereas unique genic and intergenic loci do not. Further characterization of diRNAs emerging from the 28S locus reveals the existence of two diRNA subtypes. Surprisingly, Drosha and its partner DGCR8 are dispensable for diRNA production and only one diRNAs subtype depends on Dicer processing. Furthermore, we provide evidence that diRNAs are incorporated into Argonaute. Our findings provide direct evidence for diRNA production at endogenous loci in mammalian cells and give insights into RNA processing at DSBs.
Asunto(s)
ARN Helicasas DEAD-box/genética , Reparación del ADN , ADN Intergénico/genética , ADN/genética , Endodesoxirribonucleasas/genética , ARN/genética , Ribonucleasa III/genética , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Línea Celular , ARN Helicasas DEAD-box/metabolismo , ADN/metabolismo , Roturas del ADN de Doble Cadena , Daño del ADN , ADN Intergénico/metabolismo , Endodesoxirribonucleasas/metabolismo , Sitios Genéticos , Células HeLa , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleasa III/metabolismoRESUMEN
The exosome acts on different RNA substrates and plays important roles in RNA metabolism. The fact that short non-coding RNAs are involved in the DNA damage response led us to investigate whether the exosome factor RRP6 of Drosophila melanogaster and its human ortholog EXOSC10 play a role in DNA repair. Here, we show that RRP6 and EXOSC10 are recruited to DNA double-strand breaks (DSBs) in S2 cells and HeLa cells, respectively. Depletion of RRP6/EXOSC10 does not interfere with the phosphorylation of the histone variant H2Av (Drosophila) or H2AX (humans), but impairs the recruitment of the homologous recombination factor RAD51 to the damaged sites, without affecting RAD51 levels. The recruitment of RAD51 to DSBs in S2 cells is also inhibited by overexpression of RRP6-Y361A-V5, a catalytically inactive RRP6 mutant. Furthermore, cells depleted of RRP6 or EXOSC10 are more sensitive to radiation, which is consistent with RRP6/EXOSC10 playing a role in DNA repair. RRP6/EXOSC10 can be co-immunoprecipitated with RAD51, which links RRP6/EXOSC10 to the homologous recombination pathway. Taken together, our results suggest that the ribonucleolytic activity of RRP6/EXOSC10 is required for the recruitment of RAD51 to DSBs.