title: Genome-wide analysis of novel splice variants induced by topoisomerase I poisoning shows preferential occurrence in genes encoding splicing factors.
abstract: RNA splicing is required to remove introns from pre-mRNA, and alternative splicing generates protein diversity. Topoisomerase I (Top1) has been shown to be coupled with splicing by regulating serine/arginine-rich splicing proteins. Prior studies on isolated genes also showed that Top1 poisoning by camptothecin (CPT), which traps Top1 cleavage complexes (Top1cc), can alter RNA splicing. Here, we tested the effect of Top1 inhibition on splicing at the genome-wide level in human colon carcinoma HCT116 and breast carcinoma MCF7 cells. The RNA of HCT116 cells treated with CPT for various times was analyzed with ExonHit Human Splice Array. Unlike other exon array platforms, the ExonHit arrays include junction probes that allow the detection of splice variants with high sensitivity and specificity. We report that CPT treatment preferentially affects the splicing of splicing-related factors, such as RBM8A, and generates transcripts coding for inactive proteins lacking key functional domains. The splicing alterations induced by CPT are not observed with cisplatin or vinblastine and are not simply due to reduced Top1 activity, as Top1 downregulation by short interfering RNA did not alter splicing like CPT treatment. Inhibition of RNA polymerase II (Pol II) hyperphosphorylation by 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) blocked the splicing alteration induced by CPT, which suggests that the rapid Pol II hyperphosphorylation induced by CPT interferes with normal splicing. The preferential effect of CPT on genes encoding splicing factors may explain the abnormal splicing of a large number of genes in response to Top1cc.
authors.affiliation: Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892-4255, USA.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Solier
authors.forename: Jennifer
authors.initials: J
authors.lastname: Barb
authors.forename: Barry R
authors.initials: BR
authors.lastname: Zeeberg
authors.forename: Sudhir
authors.initials: S
authors.lastname: Varma
authors.forename: Mike C
authors.initials: MC
authors.lastname: Ryan
authors.forename: Kurt W
authors.initials: KW
authors.lastname: Kohn
authors.forename: John N
authors.initials: JN
authors.lastname: Weinstein
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Munson
authors.forename: Yves
authors.initials: Y
authors.lastname: Pommier
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects / Q000235:genetics; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D002166:Camptothecin / Q000494:pharmacology; D002945:Cisplatin / Q000494:pharmacology; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000235:genetics; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000506:poisoning*; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects; D005091:Exons / Q000187:drug effects / Q000235:genetics; D014644:Genetic Variation; D055106:Genome-Wide Association Study / Q000379:methods*; D006801:Humans; D015233:Models, Statistical; D010766:Phosphorylation; D012319:RNA Polymerase II / Q000187:drug effects / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics; D034741:RNA, Small Interfering / Q000187:drug effects / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014747:Vinblastine / Q000494:pharmacology
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; D014747:Vinblastine; D012319:RNA Polymerase II; D004264:DNA Topoisomerases, Type I; D002945:Cisplatin; D002166:Camptothecin
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