HNRNPH1 Dependent Processing of EWS-FLI1 and EWSR1

Previously, we used PCR-based analysis or short-read sequencing to demonstrate that the RNA binding protein HNRNPH1 mediates the exclusion of EWSR1-exon 8 from EWS-FLI1 transcripts expressed in cell lines harboring EWSR1-intron 8 breakpoints. However, using these methods, we could not fully determine if HNRNPH1 regulates other aspects of the processing of EWS-FLI1 fusion transcripts or whether it functions in the biogenesis of EWSR1 transcripts because of the challenge of distinguishing five prime EWSR1 and EWS-FLI1 specific events. To overcome these limitations, in this study we employed PacBio long-read sequencing of EWS-FLI1 and EWSR1 transcripts following depletion of HNRNPH1 or EWS-FLI1 in TC-32 Ewing sarcoma cells.