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hnRNP A1 contacts exon 5 to promote exon 6 inclusion of apoptotic Fas gene

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Abstract

Fas is a transmembrane cell surface protein recognized by Fas ligand (FasL). When FasL binds to Fas, the target cells undergo apoptosis. A soluble Fas molecule that lacks the transmembrane domain is produced from skipping of exon 6 encoding this region in alternative splicing procedure. The soluble Fas molecule has the opposite function of intact Fas molecule, protecting cells from apoptosis. Here we show that knockdown of hnRNP A1 promotes exon 6 skipping of Fas pre-mRNA, whereas overexpression of hnRNP A1 reduces exon 6 skipping. Based on the bioinformatics approach, we have hypothesized that hnRNP A1 functions through interrupting 5′ splice site selection of exon 5 by interacting with its potential binding site close to 5′ splice site of exon 5. Consistent with our hypothesis, we demonstrate that mutations of the hnRNP A1 binding site on exon 5 disrupted the effects of hnRNP A1 on exon 6 inclusion. RNA pull-down assay and then western blot analysis with hnRNP A1 antibody prove that hnRNP A1 contacts the potential binding site RNA sequence on exon 5 but not the mutant sequence. In addition, we show that the mutation of 5′ splice site on exon 5 to a less conserved sequence destructed the effects of hnRNP A1 on exon 6 inclusion. Therefore we conclude that hnRNP A1 interacts with exon 5 to promote distal exon 6 inclusion of Fas pre-mRNA. Our study reveals a novel alternative splicing mechanism of Fas pre-mRNA.

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Acknowledgments

We thank Juan Valcarcel for providing the mutant fas minigene plasmids (e1, E23, 6-6, 5-5). This work was supported by Mid-career Researcher Program through a National Research Foundation (NRF) grant (2011-0000188 and 20120005340) funded by the Ministry of Education, Science, and Technology (MEST), Korea; and a Systems Biology Infrastructure Establishment grant provided by Gwangju Institute of Science and Technology (GIST) in 2012.

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Authors and Affiliations

  1. School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, 500-712, South Korea

    Hyun kyung Oh, Eunkyung Lee, Ha Na Jang, Jaehoon Lee, Heegyum Moon, Youngsoo Jun, Tiing Jen Loh, Sunghee Cho, Xuexiu Zheng & Haihong Shen

  2. Virginia Tech Carilion Research Institute and the Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA

    Zhi Sheng

  3. Nantong University, Nantong, China

    Jianhua Zhou

  4. iBioinfo Group, Lexington, MA, USA

    Jianhua Zhou

  5. Howard Hughes Medical Institute and Programs in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, 01605, USA

    Michael R. Green

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  1. Hyun kyung Oh
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Correspondence to Haihong Shen.

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Hyunkyung Oh and Eunkyung Lee have contributed equally to this study.

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Oh, H.k., Lee, E., Jang, H.N. et al. hnRNP A1 contacts exon 5 to promote exon 6 inclusion of apoptotic Fas gene. Apoptosis 18, 825–835 (2013). https://doi.org/10.1007/s10495-013-0824-8

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  • DOI: https://doi.org/10.1007/s10495-013-0824-8

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