The synthesis and processing of
mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final
mRNA composition varies based on
proteins that modulate splice site selection.
EWS-FLI1 is an
Ewing sarcoma (ES)
oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of
EWS-FLI1 on posttranscriptional gene regulation using both exon array and
RNA-seq. Genes that potentially regulate
oncogenesis, including CLK1,
CASP3, PPFIBP1, and TERT, validate as alternatively spliced by
EWS-FLI1. In a
CLIP-seq experiment, we find that
EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron-exon boundaries.
EWS-FLI1 also alters splicing by directly binding to known
splicing factors including DDX5,
hnRNP K, and PRPF6. Reduction of
EWS-FLI1 produces an
isoform of γ-TERT that has increased
telomerase activity compared with wild-type (WT) TERT. The small molecule
YK-4-279 is an inhibitor of
EWS-FLI1 oncogenic function that disrupts specific
protein interactions, including helicases DDX5 and
RNA helicase A (RHA) that alters RNA-splicing ratios. As such,
YK-4-279 validates the splicing mechanism of
EWS-FLI1, showing alternatively
spliced gene patterns that significantly overlap with
EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar
isoform expression patterns to cell line models expressing
EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by
EWS-FLI1.
EWS-FLI1 modulation of
mRNA splicing may provide insight into the contribution of splicing toward
oncogenesis, and, reciprocally,
EWS-FLI1 interactions with splicing
proteins may inform the splicing code.