The RNA binding proteins hnRNP H and F regulate splicing of a MYC dependent HRAS exon in Prostate Cancer Cells

The Myc proto-oncogene contributes to the pathogenesis of more than half of human cancers. Malignant transformation by Myc transcriptionally upregulates the core pre-mRNA splicing machinery and causes mis-regulation of alternative splicing. However, our understanding of how splicing changes are directed by Myc is limited. We performed a signaling pathway-guided splicing analysis to identify Myc dependent splicing events. These included an HRAS cassette exon repressed by Myc across multiple tumor types. To molecularly dissect the regulation of this HRAS exon, we used antisense oligonucleotide tiling to identify splicing enhancers and silencers in its flanking introns. RNA binding motif prediction indicated multiple binding sites for hnRNP H and hnRNP F within these cis-regulatory elements. Using siRNA knockdown and cDNA expression, we found that both hnRNP H and F activate the HRAS cassette exon. Mutagenesis and targeted RNA immunoprecipitation implicate two downstream G-rich elements in this splicing activation. Analyses of ENCODE RNA-seq datasets confirmed hnRNP H regulation of HRAS splicing. Analyses of RNA-seq datasets across multiple cancers showed a negative correlation of hnRNP H gene expression with Myc hallmark enrichment, consistent with the effect of hnRNP H on HRAS splicing. Interestingly, hnRNP F expression showed a positive correlation with Myc hallmarks and thus was not consistent with the observed effects of hnRNP F. Loss of hnRNP H/F altered cell cycle progression and induced apoptosis in the PC3 prostate cancer cell line. Collectively, our results reveal new mechanisms for Myc-dependent regulation of splicing, and point to new possible therapeutic targets in prostate cancers. ### Competing Interest Statement O.N.W currently has consulting, equity, and/or board relationships with Trethera Corporation, Kronos Biosciences, Sofie Biosciences, Breakthrough Properties, Vida Ventures, Nammi Therapeutics, Two River, Iconovir, Appia BioSciences, Neogene Therapeutics, 76Bio, and Allogene Therapeutics. Y.X. and D.L.B. have equity and serve on the board of directors for Panorama Medicine. None of these companies contributed to or directed any of the research reported in this article.