Genome-wide Analysis of Novel Splice Variants Induced by Topoisomerase I Poisoning Shows Preferential Occurrence in Genes Encoding Splicing Factors

Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892-4255, USA.
Cancer Research (Impact Factor: 9.33). 10/2010; 70(20):8055-65. DOI: 10.1158/0008-5472.CAN-10-2491
Source: PubMed


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.

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Available from: Jennifer Barb, Feb 11, 2014
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    • "In both cases, differential recruitment of elongation factors can also be involved. However, several studies pointed to an integrative model for kinetic coupling of splicing and transcription, where similar elongation changes could promote different splicing outcomes (Dutertre et al., 2010; Ip et al., 2011; Muñ oz et al., 2009; Solier et al., 2010). These genome-wide or multiple alternative splicing event (ASE) analyses revealed that, contrary to the simplest interpretation of the first come, first served mechanism (i.e., slow elongation causing high exon inclusion), a substantial part of the modified ASEs displayed an increase in exon skipping. "
    Dataset: Mol Cell

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    • "Several splicing-sensitive microarray studies have examined the effect of CPT on cotranscriptional AS [54–56]. CPT appears to reduce RNA polymerase elongation rate promoting predominantly exon inclusion [54, 55]. Interestingly, many AS events leading to exon inclusion result in the production of mRNAs containing premature stop codons (PTCs) that will undergo nonsense-mediated decay. "
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    • "Finally, a genome-wide approach was applied by Yves Pommier and colleagues to study the effect of the Topo I inhibitor camptothecin (CPT) on splicing decisions in human colon carcinoma HCT116 and breast carcinoma MCF7 cells. CPT preferentially affects splicing of transcripts for splicing factors, such as RBM8A, which belongs to the protein complex that tags exon–exon junctions after the splicing reaction (Solier et al., 2010). Interestingly, they showed that the production of the Topo I–DNA cleavage complex – Top1cc – triggered by CPT slows down RNA elongation through the rapid hyperphosphorylation of RNAPII and affects splicing profiles. "
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