title: Genotoxic stress inhibits Ewing sarcoma cell growth by modulating alternative pre-mRNA processing of the RNA helicase DHX9.
abstract: Alternative splicing plays a key role in the DNA damage response and in cancer. Ewing Sarcomas (ES) are aggressive tumors caused by different chromosomal translocations that yield in-frame fusion proteins driving transformation. RNA profiling reveals genes differentially regulated by UV light irradiation in two ES cell lines exhibiting different sensitivity to genotoxic stress. In particular, irradiation induces a new isoform of the RNA helicase DHX9 in the more sensitive SK-N-MC cells, which is targeted to nonsense-mediated decay (NMD), causing its downregulation. DHX9 protein forms a complex with RNA polymerase II (RNAPII) and EWS-FLI1 to enhance transcription. Silencing of DHX9 in ES cells sensitizes them to UV treatment and impairs recruitment of EWS-FLI1 to target genes, whereas DHX9 overexpression protects ES cells from genotoxic stress. Mechanistically, we found that UV light irradiation leads to enhanced phosphorylation and decreased processivity of RNAPII in SK-N-MC cells, which in turn causes inclusion of DHX9 exon 6A. A similar effect on DHX9 splicing was also elicited by treatment with the chemotherapeutic drug etoposide, indicating a more general mechanism of regulation in response to DNA damage. Our data identify a new NMD-linked splicing event in DHX9 with impact on EWS-FLI1 oncogenic activity and ES cell viability.
authors.affiliation: Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", Rome, Italy.|Laboratories of Cellular and Molecular Neurobiology and of Neuroimmunology, CERC, Fondazione Santa Lucia, Rome, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Fidaleo
authors.affiliation: Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", Rome, Italy.|Laboratories of Cellular and Molecular Neurobiology and of Neuroimmunology, CERC, Fondazione Santa Lucia, Rome, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Svetoni
authors.affiliation: Laboratories of Cellular and Molecular Neurobiology and of Neuroimmunology, CERC, Fondazione Santa Lucia, Rome, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Volpe
authors.affiliation: Centre de Regulació Genòmica, Barcelona, Spain.|Universitat Pompeu Fabra, Barcelona, Spain.
authors.forename: Belén
authors.initials: B
authors.lastname: Miñana
authors.affiliation: Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", Rome, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Caporossi
authors.affiliation: Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", Rome, Italy.|Laboratories of Cellular and Molecular Neurobiology and of Neuroimmunology, CERC, Fondazione Santa Lucia, Rome, Italy.
authors.forename: Maria Paola
authors.initials: MP
authors.lastname: Paronetto
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects / Q000528:radiation effects; D001859:Bone Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects / Q000528:radiation effects; D002470:Cell Survival; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D005047:Etoposide / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D015514:Oncogene Proteins, Fusion / Q000378:metabolism; D010766:Phosphorylation; D051789:Proto-Oncogene Protein c-fli-1 / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D034802:RNA-Binding Protein EWS / Q000378:metabolism; D012512:Sarcoma, Ewing / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D015398:Signal Transduction; D013997:Time Factors; D059005:Topoisomerase II Inhibitors / Q000494:pharmacology; D014162:Transfection; D014466:Ultraviolet Rays
chemical_list: C106031:EWS-FLI fusion protein; D009363:Neoplasm Proteins; D015514:Oncogene Proteins, Fusion; D051789:Proto-Oncogene Protein c-fli-1; D012322:RNA Precursors; D012333:RNA, Messenger; D034802:RNA-Binding Protein EWS; D059005:Topoisomerase II Inhibitors; D005047:Etoposide; D012319:RNA Polymerase II; C098487:DHX9 protein, human; D053487:DEAD-box RNA Helicases
keywords: DHX9; DNA damage; Ewing sarcoma; alternative splicing