title: Splicing factor SRSF1 negatively regulates alternative splicing of MDM2 under damage.
abstract: Genotoxic stress induces alternative splicing of the oncogene MDM2 generating MDM2-ALT1, an isoform attributed with tumorigenic properties. However, the mechanisms underlying this event remain unclear. Here we explore MDM2 splicing regulation by utilizing a novel minigene that mimics endogenous MDM2 splicing in response to UV and cisplatinum-induced DNA damage. We report that exon 11 is necessary and sufficient for the damage-specific alternative splicing of the MDM2 minigene and that the splicing factor SRSF1 binds exon 11 at evolutionarily conserved sites. Interestingly, mutations disrupting this interaction proved sufficient to abolish the stress-induced alternative splicing of the MDM2 minigene. Furthermore, SRSF1 overexpression promoted exclusion of exon 11, while its siRNA-mediated knockdown prevented the stress-induced alternative splicing of endogenous MDM2. Additionally, we observed elevated SRSF1 levels under stress and in tumors correlating with the expression of MDM2-ALT1. Notably, we demonstrate that MDM2-ALT1 splicing can be blocked by targeting SRSF1 sites on exon 11 using antisense oligonucleotides. These results present conclusive evidence supporting a negative role for SRSF1 in MDM2 alternative splicing. Importantly, we define for the first time, a clear-cut mechanism for the regulation of damage-induced MDM2 splicing and present potential strategies for manipulating MDM2 expression via splicing modulation.
authors.affiliation: Department of Pediatrics, The Ohio State University, Columbus, OH 43210, USA Center for Childhood Cancer, The Research Institute at Nationwide Children's Hospital, 700 Childrens Drive WA5023, Columbus, OH 43205, USA.
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Comiskey
authors.affiliation: Department of Pediatrics, The Ohio State University, Columbus, OH 43210, USA Center for Childhood Cancer, The Research Institute at Nationwide Children's Hospital, 700 Childrens Drive WA5023, Columbus, OH 43205, USA.
authors.forename: Aishwarya G
authors.initials: AG
authors.lastname: Jacob
authors.affiliation: Department of Pediatrics, The Ohio State University, Columbus, OH 43210, USA Center for Childhood Cancer, The Research Institute at Nationwide Children's Hospital, 700 Childrens Drive WA5023, Columbus, OH 43205, USA.
authors.forename: Ravi K
authors.initials: RK
authors.lastname: Singh
authors.affiliation: Department of Pediatrics, The Ohio State University, Columbus, OH 43210, USA Center for Childhood Cancer, The Research Institute at Nationwide Children's Hospital, 700 Childrens Drive WA5023, Columbus, OH 43205, USA.
authors.forename: Aixa S
authors.initials: AS
authors.lastname: Tapia-Santos
authors.affiliation: Department of Pediatrics, The Ohio State University, Columbus, OH 43210, USA Center for Childhood Cancer, The Research Institute at Nationwide Children's Hospital, 700 Childrens Drive WA5023, Columbus, OH 43205, USA dawn.chandler@nationwidechildrens.org.
authors.forename: Dawn S
authors.initials: DS
authors.lastname: Chandler
mesh_terms: D017398:Alternative Splicing; D004249:DNA Damage; D005091:Exons; D006367:HeLa Cells; D006801:Humans; D061986:MCF-7 Cells; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D020319:Oligodeoxyribonucleotides, Antisense; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D038621:Regulatory Sequences, Ribonucleic Acid; D012208:Rhabdomyosarcoma / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D013312:Stress, Physiological / Q000235:genetics
chemical_list: D009687:Nuclear Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D016601:RNA-Binding Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; D000068103:Serine-Arginine Splicing Factors; D051736:Proto-Oncogene Proteins c-mdm2
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