Post-transcriptional and co-transcriptional regulation of RNA processing play a crucial role in generating protein diversity, thus modulating many cellular processes and responses to external stimuli. A pivotal role in this fine regulation is played by RNA binding proteins. The Ewing sarcoma protein EWS belongs to the TET family (FUS/TLS, EWS, TAF15) of proteins. They bind RNA as well as DNA, and are implicated in RNA transcription, splicing, transport, as well as in intracellular signaling and in maintenance of genomic integrity. It was shown that EWS interacts with U1C, SF1 and several SR proteins and hnRNPs. However, its implication in constitutive or alternative splicing is still largely unknown. In order to identify in vivo RNA targets of EWS, we performed cross linked in vivo immunoprecipitation in Hela cells, followed by high-throughput sequencing of the isolated RNAs (HITS-CLIP). By comparing EWS and control IgG immunoprecipitates, we obtained 467818 reads unambiguously mapped to the genome, forming 66753 clusters. We identified 17859 clusters overlapping 8233 different genes. The identified clusters were enriched in exons, revealing an increased density at 3’ and 5’ splice site and highlighting a potential role of EWS in splicing regulation. In addition, a core motif search identified a consensus sequence for EWS binding with high G content, thus offering the possibility to build an RNA map of EWS regulation. Gene ontology analysis of EWS target genes displaying clusters upregulated at least five times versul the control IgG, revealed an enrichment of genes involved in DNA recombination and repair and MAPK signaling pathways, highlighting a potential role in the DNA damage response. Remarkably, EWS knockout mice displayed hypersensitivity to ionizing radiation, demonstrating a role for EWS in the protection of the genome integrity and in the response to genotoxic stress. Moreover, we performed a microarray analysis of the splicing events changed upon EWS knockdown, using a exon junction array platform with 1804 alternative splicing events in 482 genes related to cancer and RNA processing, and we found that 80% of the genes that displayed AS changes upon EWS knockdown contained clip tags in our CLIP-HITS experiment. Strikingly, gene ontology analysis of the AS target genes indicated that they are also involved in MAPK and ATM signaling pathways, which are activated by external stress. Our results identify a new role for EWS in mediating the DNA damage response through the regulation of AS choices and RNA processing.
Identification of in vivo RNA targets of the Ewing Sarcoma protein EWS by HITS-CLIP
Paronetto MP;
2010-01-01
Abstract
Post-transcriptional and co-transcriptional regulation of RNA processing play a crucial role in generating protein diversity, thus modulating many cellular processes and responses to external stimuli. A pivotal role in this fine regulation is played by RNA binding proteins. The Ewing sarcoma protein EWS belongs to the TET family (FUS/TLS, EWS, TAF15) of proteins. They bind RNA as well as DNA, and are implicated in RNA transcription, splicing, transport, as well as in intracellular signaling and in maintenance of genomic integrity. It was shown that EWS interacts with U1C, SF1 and several SR proteins and hnRNPs. However, its implication in constitutive or alternative splicing is still largely unknown. In order to identify in vivo RNA targets of EWS, we performed cross linked in vivo immunoprecipitation in Hela cells, followed by high-throughput sequencing of the isolated RNAs (HITS-CLIP). By comparing EWS and control IgG immunoprecipitates, we obtained 467818 reads unambiguously mapped to the genome, forming 66753 clusters. We identified 17859 clusters overlapping 8233 different genes. The identified clusters were enriched in exons, revealing an increased density at 3’ and 5’ splice site and highlighting a potential role of EWS in splicing regulation. In addition, a core motif search identified a consensus sequence for EWS binding with high G content, thus offering the possibility to build an RNA map of EWS regulation. Gene ontology analysis of EWS target genes displaying clusters upregulated at least five times versul the control IgG, revealed an enrichment of genes involved in DNA recombination and repair and MAPK signaling pathways, highlighting a potential role in the DNA damage response. Remarkably, EWS knockout mice displayed hypersensitivity to ionizing radiation, demonstrating a role for EWS in the protection of the genome integrity and in the response to genotoxic stress. Moreover, we performed a microarray analysis of the splicing events changed upon EWS knockdown, using a exon junction array platform with 1804 alternative splicing events in 482 genes related to cancer and RNA processing, and we found that 80% of the genes that displayed AS changes upon EWS knockdown contained clip tags in our CLIP-HITS experiment. Strikingly, gene ontology analysis of the AS target genes indicated that they are also involved in MAPK and ATM signaling pathways, which are activated by external stress. Our results identify a new role for EWS in mediating the DNA damage response through the regulation of AS choices and RNA processing.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
