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

Xinyuan Chen, Harry Taegyun Yang, Beatrice Zhang, John W. Phillips, Donghui Cheng, Frank Rigo, Owen N. Witte, Yi Xing, Douglas L. Black
doi: https://doi.org/10.1101/2022.11.29.518269
Xinyuan Chen
1Molecular Biology Interdepartmental Doctoral Program, University of California, Los Angeles, Los Angeles, California 90095, USA
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Harry Taegyun Yang
2Bioinformatics Interdepartmental Graduate Program, University of California, Los Angeles, California 90095, USA
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Beatrice Zhang
3Center for Computational and Genomic Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA
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John W. Phillips
4Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095, USA
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Donghui Cheng
4Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095, USA
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Frank Rigo
5Ionis Pharmaceuticals, Inc., 2855 Gazelle Ct., Carlsbad, California 92010, USA
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Owen N. Witte
4Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095, USA
6Department of Molecular and Medical Pharmacology, University of California, Los Angeles, California 90095, USA
7Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California 90095, USA
8Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, California 90095, USA.
9Molecular Biology Institute, University of California, Los Angeles, California 90095, USA
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Yi Xing
3Center for Computational and Genomic Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA
10Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
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  • For correspondence: dougb@microbio.ucla.edu xingyi@chop.edu
Douglas L. Black
4Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095, USA
7Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California 90095, USA
8Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, California 90095, USA.
9Molecular Biology Institute, University of California, Los Angeles, California 90095, USA
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  • For correspondence: dougb@microbio.ucla.edu xingyi@chop.edu
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ABSTRACT

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.

SIGNIFICANCE STATMENT Myc Transformation by the proto-oncogene c-Myc causes dysregulation of the pre-mRNA splicing reaction in cancer, but it is not known how mRNA isoform changes are directed by Myc. Here, we use bioinformatics to identify a splicing event in another proto-oncogene, HRAS, that is regulated by Myc across multiple tumor types. We identify new splicing regulators, hnRNP’s H and F, that control this HRAS exon by binding to enhancer elements within its downstream intron. Additional pan-cancer bioinformatic analyses show hnRNP H expression to be anti- correlated with Myc hallmarks, consistent with the reduced splicing of the HRAS exon in Myc driven cancer. These findings uncover new mechanisms by which Myc can alter splicing in cancer cells and provide new molecular targets for potential therapeutics.

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.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted November 29, 2022.
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The RNA binding proteins hnRNP H and F regulate splicing of a MYC dependent HRAS exon in Prostate Cancer Cells
Xinyuan Chen, Harry Taegyun Yang, Beatrice Zhang, John W. Phillips, Donghui Cheng, Frank Rigo, Owen N. Witte, Yi Xing, Douglas L. Black
bioRxiv 2022.11.29.518269; doi: https://doi.org/10.1101/2022.11.29.518269
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The RNA binding proteins hnRNP H and F regulate splicing of a MYC dependent HRAS exon in Prostate Cancer Cells
Xinyuan Chen, Harry Taegyun Yang, Beatrice Zhang, John W. Phillips, Donghui Cheng, Frank Rigo, Owen N. Witte, Yi Xing, Douglas L. Black
bioRxiv 2022.11.29.518269; doi: https://doi.org/10.1101/2022.11.29.518269

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