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.

    • "The AS of many RBPs and splicing factors was altered by DNA damage, including SRSF1, RBM8A, ZRANB2, SF3B3, TIA-1, and TIAL1, among others. Interestingly, in some of these cases, included exons introduced premature termination codons that could elicit nonsense-mediated mRNA decay [78,80,109]. The hSlu7 splicing factor has been suggested to hold the 5′ exon within the spliceosome complex while the correct 3' splice site is selected. "
    [Show abstract] [Hide abstract] ABSTRACT: Multicellular organisms must ensure genome integrity to prevent accumulation of mutations, cell death, and cancer. The DNA damage response (DDR) is a complex network that senses, signals and executes multiple programs including DNA repair, cell cycle arrest, senescence and apoptosis. This entails regulation of a variety of cellular processes: DNA replication and transcription, RNA processing, mRNA translation and turnover, and post-translational modification, degradation and relocalization of proteins. Accumulated evidence over the past decades has shown that RNAs and RNA metabolism are both regulators and regulated actors of the DDR. This review aims to present a comprehensive overview of the current knowledge on the many interactions between the DNA damage and RNA fields.
    No preview · Article · Mar 2016 · Journal of Molecular Biology
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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
    Full-text · Dataset · Oct 2015
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    • "Moreover, MCF7 cells are well-characterized notably in terms of their response to chemotherapeutic drugs. Our results indicate that cisplatin affects the expression level (absolute fold change >2) of more than 500 genes and provokes changes in at least 700 splicing events, thereby extending previous observations that chemotherapeutic agents affect AS [6,13,16]. This splicing reprogramming also occurs in other transformed cell lines including the breast cancer cell lines MDA-MB-231 and BT549, the endometrial adenocarcinoma cell line Ishikawa and in primary fibroblasts. "
    Full-text · Dataset · Apr 2015
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