Post-transcriptional and co-transcriptional regulation of RNA processing plays a crucial role in generating protein diversity, thus modulating many cellular processes and responses to external stimuli. RNA binding proteins play a pivotal role in this fine regulation, by binding to cis-regulatory elements (enhancers and silencers), and helping the recruitment of the spliceosome. 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 reported that EWS interacts with U1C, SF1 and several SR proteins and hnRNPs. More recently, we and othershave documented that EWS modulates alternative splicing of genes involved in the DNA damage response(DDR), including key regulators of genotoxic stress. However, its function in constitutive or alternative splicing is still largely unknown. In order to identify in vivo RNA targets of EWS, we performed in vivo cross-linking and immunoprecipitation followed by high-throughput sequencing of the isolated RNAs (HITS-CLIP) in HeLa cells. We obtained 467818 reads unambiguously mapped to the genome, forming 97011 clusters outside repeats. From these, we identified 20776 clusters overlapping 8898 different genes. The identified clusters were enriched in exons, with increased density near the 5’ splice sites, strongly suggesting a role for EWS in splicing regulation. Moreover, a sequence motif search identified a consensus sequence for EWS binding harboring G-rich motifs, as previously suggested for TLS by SELEX approach. The most enriched cluster in EWS HITS-CLIP was found in exon 6 of FAS pre-mRNA. Using FAS as a paradigm of EWS target we demonstrated that EWS acts as a splicing activator which binds exonic enhancer elements to help in the recruitment of U1snRNP to weak 5’splice sites.
HITS-CLIP unveils a role for EWS in 5'splice site definition
Paronetto MP;
2012-01-01
Abstract
Post-transcriptional and co-transcriptional regulation of RNA processing plays a crucial role in generating protein diversity, thus modulating many cellular processes and responses to external stimuli. RNA binding proteins play a pivotal role in this fine regulation, by binding to cis-regulatory elements (enhancers and silencers), and helping the recruitment of the spliceosome. 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 reported that EWS interacts with U1C, SF1 and several SR proteins and hnRNPs. More recently, we and othershave documented that EWS modulates alternative splicing of genes involved in the DNA damage response(DDR), including key regulators of genotoxic stress. However, its function in constitutive or alternative splicing is still largely unknown. In order to identify in vivo RNA targets of EWS, we performed in vivo cross-linking and immunoprecipitation followed by high-throughput sequencing of the isolated RNAs (HITS-CLIP) in HeLa cells. We obtained 467818 reads unambiguously mapped to the genome, forming 97011 clusters outside repeats. From these, we identified 20776 clusters overlapping 8898 different genes. The identified clusters were enriched in exons, with increased density near the 5’ splice sites, strongly suggesting a role for EWS in splicing regulation. Moreover, a sequence motif search identified a consensus sequence for EWS binding harboring G-rich motifs, as previously suggested for TLS by SELEX approach. The most enriched cluster in EWS HITS-CLIP was found in exon 6 of FAS pre-mRNA. Using FAS as a paradigm of EWS target we demonstrated that EWS acts as a splicing activator which binds exonic enhancer elements to help in the recruitment of U1snRNP to weak 5’splice sites.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
