HNRNPH1-dependent splicing of a fusion oncogene reveals a targetable RNA G-quadruplex interaction

The primary oncogenic event in ~85% of Ewing sarcomas is a chromosomal translocation that generates a fusion oncogene encoding an aberrant transcription factor. The exact genomic breakpoints within the translocated genes, EWSR1 and FLI1, vary; however, in EWSR1, breakpoints typically occur within introns 7 or 8. We previously found that in Ewing sarcoma cells harboring EWSR1 intron 8 breakpoints, the RNA-binding protein heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) facilitates a splicing event that excludes EWSR1 exon 8 from the EWS-FLI1 pre-mRNA to generate an in-frame mRNA. Here, we show that the processing of distinct EWS-FLI1 pre-mRNAs by HNRNPH1, but not other homologous family members, resembles alternative splicing of transcript variants of EWSR1. We demonstrate that HNRNPH1 recruitment is driven by guanine-rich sequences within EWSR1 exon 8 that have the potential to fold into RNA G-quadruplex structures. Bioinformatic analysis of transcriptome-wide profiles for RNA G-quadruplexes and RNA targets of HNRNPH1 revealed enrichment of sequences containing two-quartet G-quadruplex configurations within exonic HNRNPH1 binding sites. Critically, we demonstrate that a synthetic RNA mimic of one of these two-quartet G-quadruplexes modulates HNRNPH1 binding and induces a concentration-dependent decrease in the growth of an EWSR1 exon 8 fusion-positive Ewing sarcoma cell line. Finally, we show that EWSR1 exon 8 fusion-positive Ewing sarcoma cell lines are more sensitive to treatment with the pan-quadruplex binding molecule, pyridostatin (PDS), than EWSR1 exon 8 fusion-negative lines. In vitro binding studies reveal that PDS modulates HNRNPH1 binding to EWSR1 exon 8. Also, the treatment of EWSR1 exon 8 fusion-positive Ewing sarcoma cells with PDS decreases EWS-FLI1 transcriptional activity, reversing the transcriptional deregulation driven by EWS-FLI1. Our findings illustrate that modulation of the alternative splicing of EWS-FLI1 pre-mRNA is a novel strategy for future therapeutics against the EWSR1 exon 8 containing fusion oncogenes present in a third of Ewing sarcoma. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

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