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. Cancer Res; 70(20); 8055-65. ©2010 AACR.

A recently evolved class of alternative 3′-terminal exons involved in cell cycle regulation by topoisomerase inhibitors

“…As expected from previous studies and from its ability to inhibit transcription elongation [20][21][22] , CPT favored inclusion more often than exclusion of cassette exons (Fig. 1c).…”

“…Thus, cell responses to DNA damage may involve the coordinated regulation of both nuclear and cytoplasmic sets of transcripts by HuR. Large-scale regulation of alternative polyadenylation and splicing are increasingly involved in cellular processes, including proliferation and genotoxic responses, respectively [1][2][3][4][5][6][7][8]16,[19][20][21] . In this context, our data provide several advances.…”

“…Several genes involved in the DNA damage response, such as MDM2 that encodes the main repressor of p53, are regulated at the splicing level in response to genotoxic agents 16 . Recently, UV irradiation and the topoisomerase (TOP) I inhibitor, camptothecin (CPT), were shown to increase the inclusion of many cassette exons through an inhibition of transcription elongation, which gives more time for the cotranscriptional recognition and inclusion of alternative exons [19][20][21] . CPT also induces skipping of a set of exons, in part by disrupting the interaction between the EWS and YB-1 proteins 22 .…”

The emerging role of pre-messenger RNA splicing in stress responses: Sending alternative messages and silent messengers

“…21 Likewise, several recent studies identified hundreds of splice variants regulated by CPT. 29,37,43 A kinetic study revealed both early and late events, with a majority of events regulated at later time points (e.g., 15-20 h). 43 GO term analysis of the corresponding genes revealed enrichment in functions related to RNA processing, mitosis and apoptosis.…”

The Natural Inhibitor of DNA Topoisomerase I, Camptothecin, Modulates HIF-1α Activity by Changing miR Expression Patterns in Human Cancer Cells

“…In the past few years, the attention has been oriented back on these chemical inhibitors as several additional effects have been highlighted, mostly reflected on the interference with transcription. We evidenced that Top1 inhibition favors RNA Pol II escape from the promoter-proximal pausing site of several genes, enhances histone acetylation and reduces nucleosome density in a CDK-dependent manner, and finally induces alterations of splicing events (9,14,23,28,29,32). It is noteworthy that camptothecin can increase genome-wide the level of antisense RNAs at CpG island promoters of divergent genes (30) and, specifically for the present work, it is able to induce the transcription of long noncoding antisense RNAs at the 5 0 and 3 0 end of the HIF-1a gene (23,24), which may regulate gene expression posttranscriptionally (24).…”