Role of EWS/EWS-FLI1 in post-transcriptional gene regulation

TET (TLS, EWS, TAF15) genes encode an evolutionary conserved family of RNA binding proteins which are frequently rearranged with transcription factor genes in sarcomas and acute leukemias. TET proteins are involved in RNA processing but oncogenic TET-fusion proteins are considered aberrant transcriptional regulators. The first identified member of the TET family is EWS which is fused to the ETS transcription factor FLI1 as a result of chromosomal translocation in Ewings sarcoma family tumors (ESFT). So far, investigations were primarily directed to target gene discovery and the DNA-binding and transcriptional regulation function of TET-fusion proteins. Since EWS-FLI1 may affect post-transcriptional RNA metabolism either directly or through interference with normal TET function., and increasing attention is payed to abnormal RNA processing in tumorigenesis, we propose to study the role of EWS and EWS-FLI1 in processing of mRNA and microRNA precursors. Several pieces of evidence support a role for EWS-FLI1 in posttranscriptional RNA metabolism: i) The minimal transforming domains of EWS-FLI1 do not fully overlap with its transcriptional activation and DNA-binding domains; ii) EWS and EWS-FLI1 associate not only with components of RNA polymerase II but also with various RNA processing factors; iii) EWS-FLI1 can physically interact with EWS; iv) There is evidence that EWS-FLI1 can interfere with EWS mediated splicing; v) EWS associates with the primary microRNA precursor-processing RNaseIII, DROSHA; We hypothesize that EWS-FLI1 could play a key role in tumor associated post-transcriptional changes such as aberrant splicing or microRNA processing. The experimental strategies chosen in the present proposal are based on Ewings sarcoma cell line models and tools previously established in our laboratory. A comprehensive structure- function analysis employing site directed mutagenesis and subsequent expression in a unique EWS-deficient Ewing s sarcoma cell line model will reveal functional interactions and dissect the mutual influence of RNA and DNA binding of EWS and EWS-FLI1 on overall mRNA and microRNA expression. As readouts, genome wide mRNA and microRNA expression profiling will be combined with readouts for RNA processing using recently developed exon splice arrays. In order to verify clinically relevant targets of EWS/EWS-FLI1 regulation, results from cell line models will be compared to expression data from primary tumors. As a candidate approach, a particular subset of previously identified genes from our laboratory carrying intronic EWS-FLI1 binding sites, will be analyzed in detail for EWS-FLI1 dependent changes in RNA processing. Additionally, we will attempt to directly isolate RNA molecules bound to EWS within the Ewings sarcoma context and define their relation to differentially expressed mRNA isoforms and microRNAs. In the present application we propose to exploit the EWS / EWS-FLI1 system as paradigm for studying RNA-related mechanisms of tumorigenesis ultimately leading to the identification of new tumor-targeting opportunities.

TET (TLS, EWS, TAF15) genes encode an evolutionary conserved family of RNA binding proteins which are frequently rearranged with transcription factor genes in sarcomas and acute leukemias. TET proteins are involved in RNA processing but oncogenic TET-fusion proteins are considered aberrant transcriptional regulators. The first identified member of the TET family is EWS which is fused to the ETS transcription factor FLI1 as a result of chromosomal translocation in Ewing`s sarcoma family tumors (ESFT). So far, investigations were primarily directed to target gene discovery and the DNA-binding and transcriptional regulation function of TET-fusion proteins. Since EWS-FLI1 may affect post-transcriptional RNA metabolism either directly or through interference with normal TET function., and increasing attention is payed to abnormal RNA processing in tumorigenesis, we propose to study the role of EWS and EWS-FLI1 in processing of mRNA and microRNA precursors. Several pieces of evidence support a role for EWS-FLI1 in posttranscriptional RNA metabolism: 1. The minimal transforming domains of EWS-FLI1 do not fully overlap with its transcriptional activation and DNA-binding domains; 2. EWS and EWS-FLI1 associate not only with components of RNA polymerase II but also with various RNA processing factors; 3. EWS-FLI1 can physically interact with EWS; 4. There is evidence that EWS-FLI1 can interfere with EWS mediated splicing; 5. EWS associates with the primary microRNA precursor-processing RNaseIII, DROSHA. We hypothesize that EWS-FLI1 could play a key role in tumor associated post-transcriptional changes such as aberrant splicing or microRNA processing. The experimental strategies chosen in the present proposal are based on Ewing`s sarcoma cell line models and tools previously established in our laboratory. A comprehensive structure- function analysis employing site directed mutagenesis and subsequent expression in a unique EWS-deficient Ewing`s sarcoma cell line model will reveal functional interactions and dissect the mutual influence of RNA and DNA binding of EWS and EWS-FLI1 on overall mRNA and microRNA expression. As readouts, genome wide mRNA and microRNA expression profiling will be combined with readouts for RNA processing using recently developed exon splice arrays. In order to verify clinically relevant targets of EWS/EWS-FLI1 regulation, results from cell line models will be compared to expression data from primary tumors. As a candidate approach, a particular subset of previously identified genes from our laboratory carrying intronic EWS-FLI1 binding sites, will be analyzed in detail for EWS-FLI1 dependent changes in RNA processing. Additionally, we will attempt to directly isolate RNA molecules bound to EWS within the Ewing`s sarcoma context and define their relation to differentially expressed mRNA isoforms and microRNAs. In the present application we propose to exploit the EWS / EWS-FLI1 system as paradigm for studying RNA-related mechanisms of tumorigenesis ultimately leading to the identification of new tumor-targeting opportunities.