Cellular Transformation by Ews/fli-1 Is Associated with Disruption of Rna Splicing

INTRODUCTION: In over 85% cases of Ewing’s sarcoma and peripheral primitive neuroectodermal tumors (PNET), a highly specific and recurrent t(11;22) balanced chromosomal translocation results in the fusion of the 5’ half of a gene known as EWS on chromosome 22 to the 3’ portion of the chromosome 11-derived gene FLI-1. The resultant EWS/FLI-1 fusion protein contains EWS-derived sequence as its N-terminal half and FLI-1-derived sequence as its C-terminal half. The EWS/FLI-1 fusion protein is thought to act as a chimeric transcription factor leading to malignant transformation via transactivation of FLI-1 target genes. Despite evidence implicating transcriptional alteration by EWS/FLI-1, recent studies from our laboratories (Jaishankar et al., Oncogene, 1999; Yang et al., J. Biol. Chem., 2000; Chansky et al., Cancer Res., 2001; Knoop et al., J. Biol. Chem., 2001) have shown that wild-type EWS interacts both with hyperphosphorylated RNA Pol II and with a family of serine-arginine(SR) splicing factors, whereas the EWS/FLI-1 fusion protein disrupts this coupling by binding to RNA Pol II but failing to recruit SR proteins due to replacement of its C-terminal domain by FLI-1. In addition, several lines of evidence suggest that alternative mechanisms may be involved in transformation. First, although EWS/FLI-1 is a more potent transcription factor than FLI-1, deletional studies indicate that the domain within EWS required for transformation differs from the domain required for transactivation. Second, a mutagenesis study demonstrated that a point mutation abolishing the DNAbinding activity of EWS/FLI-1 did not abolish its transforming ability. Third, our previous in vivo assays have shown that EWS/FLI-1 interferes with RNA splicing. Thus at least a portion of the oncogenic activity of EWS/FLI-1 is independent of transcription. Through the analysis of different EWS/FLI-1 mutants, we report here that the N-terminal 81 amino acids of EWS/FLI-1 mediate interaction with RNA Pol II. These EWS/FLI-1 mutants were also cotransfected with a luciferase reporter gene for a transcription assay, and cotransfected with an E1A reporter gene for a splicing assay. Our results showed that the transforming activity of EWS/FLI-1 is not strictly dependent upon the DNA-binding or transactivating ability of EWS/FLI-1. Instead, cellular transformation by EWS/FLI-1 is associated with its ability to interfere with RNA splicing.