title: How is Herstatin, a tumor suppressor splice variant of the oncogene HER2, regulated?
abstract: The human epidermal growth factor receptor 2 (HER2)/receptor tyrosine-protein kinasebB-2 (ERBB2) is overexpressed in 20-30% of breast tumors leading to faster growing and more aggressive tumors. Alternative splicing generates a functionally distinct HER2 variant called Herstatin, which is produced by the inclusion of intron 8. Herstatin acts as a tumor suppressor by effectively blocking HER2 activity and cell proliferation, while promoting apoptosis. In the present study we investigated HER2 pre-mRNA regulatory sequences and splicing factors which regulate the alternative splicing of Herstatin. A Herstatin minigene, comprising exon 8/intron 8/exon 9 of HER2 was generated and subsequent in vitro splicing assays revealed that RNA secondary structure and somatic mutations did not impact on inclusion of intron 8. However, using RNase-assisted RNA chromatography, followed by mass spectrometry, we identified six RNA-binding proteins (splicing factors) that bind to RNA sequences surrounding exon 8/intron 8 and intron 8/exon 9 boundaries; these included hnRNP I, H1, D, A2/B1 and hnRNPA1 plus the SR protein SRSF1. Specifically, overexpression of hnRNP A1 significantly increased retention of intron 8 resulting in higher levels of Herstatin in SKBR3 breast cancer cells whereas SRSF1 only had a marginal effect in decreasing Herstatin but increased exogenous HER2 levels under these experimental conditions. In conclusion, we have identified the first splicing factors and regulatory sequences that are involved in the production of Herstatin.
authors.affiliation: a Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University , Newcastle , UK.
authors.forename: Marco
authors.initials: M
authors.lastname: Silipo
authors.affiliation: a Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University , Newcastle , UK.
authors.forename: Hannah
authors.initials: H
authors.lastname: Gautrey
authors.affiliation: a Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University , Newcastle , UK.
authors.forename: Swapna
authors.initials: S
authors.lastname: Satam
authors.affiliation: b Northern Institute for Cancer Research, Faculty of Medical Sciences, Newcastle University , Newcastle , UK.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lennard
authors.affiliation: a Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University , Newcastle , UK.
authors.forename: Alison
authors.initials: A
authors.lastname: Tyson-Capper
mesh_terms: D017398:Alternative Splicing; D017209:Apoptosis; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D005091:Exons; D005260:Female; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D007438:Introns; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D000072260:RNA Splicing Factors / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics*; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D018719:Erb-b2 Receptor Tyrosine Kinases; D025521:Tumor Suppressor Proteins; C434665:herstatin; C508053:ERBB2 protein, human
keywords: Breast cancer; HER2 splice variants; RNA-binding proteins; oncogene HER2/erbB-2