RESUMEN
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.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Recombinación Homóloga/genética , Animales , Western Blotting , Proliferación Celular , Inmunoprecipitación de Cromatina , Proteínas de Drosophila/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Exorribonucleasas/antagonistas & inhibidores , Exorribonucleasas/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/antagonistas & inhibidores , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Células HeLa , Histonas/metabolismo , Humanos , Fosforilación , ARN Interferente Pequeño/genética , Recombinasa Rad51/metabolismoRESUMEN
U12-type introns are a rare class of introns in the genomes of diverse eukaryotes. In the human genome, they number over 700. A subset of these introns has been shown to be spliced at a slower rate compared to the major U2-type introns. This suggests a rate-limiting regulatory function for the minor spliceosome in the processing of transcripts containing U12-type introns. However, both the generality of slower splicing and the subsequent fate of partially processed pre-mRNAs remained unknown. Here, we present a global analysis of the nuclear retention of transcripts containing U12-type introns and provide evidence for the nuclear decay of such transcripts in human cells. Using SOLiD RNA sequencing technology, we find that, in normal cells, U12-type introns are on average 2-fold more retained than the surrounding U2-type introns. Furthermore, we find that knockdown of RRP41 and DIS3 subunits of the exosome stabilizes an overlapping set of U12-type introns. RRP41 knockdown leads to slower decay kinetics of U12-type introns and globally upregulates the retention of U12-type, but not U2-type, introns. Our results indicate that U12-type introns are spliced less efficiently and are targeted by the exosome. These characteristics support their role in the regulation of cellular mRNA levels.
Asunto(s)
Núcleo Celular/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Intrones , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , ARN Mensajero/metabolismo , Línea Celular , Núcleo Celular/enzimología , Complejo Multienzimático de Ribonucleasas del Exosoma/antagonistas & inhibidores , Humanos , ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidoresRESUMEN
PURPOSE: We utilized whole-genome mapping of promoters that are activated by DNA hypomethylation in hepatocellular carcinoma (HCC) clinical samples to shortlist novel targets for anticancer therapeutics. We provide a proof of principle of this approach by testing six genes short-listed in our screen for their essential role in cancer growth and invasiveness. EXPERIMENTAL DESIGN: We used siRNA- or shRNA-mediated depletion to determine whether inhibition of these genes would reduce human tumor xenograft growth in mice as well as cell viability, anchorage-independent growth, invasive capacities, and state of activity of nodal signaling pathways in liver, breast, and bladder cancer cell lines. RESULTS: Depletion of EXOSC4, RNMT, SENP6, WBSCR22, RASAL2, and NENF effectively and specifically inhibits cancer cell growth and cell invasive capacities in different types of cancer, but, remarkably, there is no effect on normal cell growth, suggesting a ubiquitous causal role for these genes in driving cancer growth and metastasis. Depletion of RASAL2 and NENF in vitro reduces their growth as explants in vivo in mice. RASAL2 and NENF depletion interferes with AKT, WNT, and MAPK signaling pathways as well as regulation of epigenetic proteins that were previously demonstrated to drive cancer growth and metastasis. CONCLUSION: Our results prove that genes that are hypomethylated and induced in tumors are candidate targets for anticancer therapeutics in multiple cancer cell types. Because these genes are particularly activated in cancer, they constitute a group of targets for specific pharmacologic inhibitors of cancer and cancer metastasis. Clin Cancer Res; 20(12); 3118-32. ©2014 AACR.