Regulation of FAS Exon Definition and Apoptosis by the Ewing Sarcoma Protein. Homo sapiens

The Ewing’s sarcoma protein EWS belongs to the TET family (FUS/TLS, EWS, TAF15) of RNA and DNA binding proteins, implicated in DNA transcription, pre-mRNA splicing and maintenance of genomic integrity. Translocations of these genes are characteristic of particular neoplasias, including Ewing’s sarcoma. To identify physiological RNA targets of EWS, we performed in vivo cross-linking and immunoprecipitation followed by high-throughput RNA sequencing (HITS-CLIP/CLIP-Seq) in HeLa cells. Sequencing identified EWS binding sites characterized by guanosine-rich motifs in nearly 9000 genes, with particular enrichment in exonic regions near 5’ splice sites. Exon 6 of the Fas/CD95 receptor, which is alternatively spliced to generate isoforms with opposing activities in programmed cell death, was found as a prominent EWS CLIP target, as well as by chromatin-immunoprecipitation (ChIP) and functional analysis. Manipulation of EWS levels and mutation of EWS binding sites led to changes in alternative splicing consistent with EWS promoting exon 6 inclusion and leading to the synthesis of the pro-apoptotic Fas/CD95 isoform. Biochemical characterization of factors associated with FAS exon 6 are consistent with the notion that EWS binds to exonic sequences near the 5’ splice site and promotes the recruitment of U1snRNP, favoring also recognition of the upstream 3' splice site by U2AF and thus exon definition. Consistent with a role for EWS in the regulation of programmed cell death, cells depleted of EWS show decreased sensitivity to Fas-induced apoptosis. We discuss the potential implications of this novel function of EWS in Ewing’s sarcoma. Overall design: CLIP-Seq analysis of EWS, with 2 biological replicates of EWS and one non-specific control

尤因肉瘤蛋白EWS（Ewing’s sarcoma protein EWS）属于RNA与DNA结合蛋白的TET家族（成员包含FUS/TLS、EWS、TAF15），该家族蛋白参与DNA转录、前体mRNA剪接以及基因组完整性维持。这类基因的易位是包括尤因肉瘤在内的特定肿瘤的标志性特征。为鉴定EWS的生理性RNA靶标，我们在海拉细胞（HeLa cells）中开展了体内交联免疫沉淀联合高通量RNA测序（HITS-CLIP/CLIP-Seq）实验。测序结果显示，EWS的结合位点以近9000个基因中的富鸟嘌呤基序为特征，且在5’剪接位点附近的外显子区域呈现显著富集。Fas/CD95受体的第6外显子可通过可变剪接产生在程序性细胞死亡中功能相反的异构体，该外显子被鉴定为EWS CLIP的关键靶标，同时也通过染色质免疫沉淀（ChIP）与功能分析得到验证。调控EWS的表达水平或突变其结合位点可引发可变剪接模式改变，该结果与EWS促进外显子6保留并进而合成促凋亡型Fas/CD95异构体的功能一致。针对Fas第6外显子相关因子的生化表征结果与下述观点一致：EWS结合至5’剪接位点附近的外显子序列，促进U1小核核糖核蛋白（U1snRNP）的招募，同时促进U2辅助因子（U2AF）识别上游3’剪接位点，进而完成外显子定义。与EWS在程序性细胞死亡调控中的作用一致，EWS表达耗竭的细胞对Fas诱导的凋亡敏感性显著降低。我们探讨了EWS的这一新功能在尤因肉瘤中的潜在意义。实验整体设计：对EWS开展CLIP-Seq分析，设置2份EWS生物学重复样本与1份非特异性对照样本。