Genome-wide analysis of novel splice variants induced by topoisomerase I poisoning shows preferential occurrence in genes encoding splicing factors

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 SR 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 impact 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 to 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 siRNA did not alter splicing like CPT treatment. Inhibition of RNA polymerase II (Pol II) hyperphosphorylation by 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.

probes, the ExonSVD model ( y ijk = μ + A' i D k + E ik + β j(i) + C k + ε ijk ) includes an explicit parameter, D, for probeset responsiveness. Also, the ExonSVD model can directly handle body and junction probes directly without further elaboration. The body probes primarily determine the differential expression while the junction probes which are sensitive to the varying levels of exon-exon junctions, are the primary source of information regarding alternative splicing. The E effect of the model (alternative splicing effect) is determined as the residual after differential expression effects are accounted for, and is tested for significance to determine whether alternative splicing has occurred. This new model alleviates the need for pre-filtering to eliminate dead and unresponsive probesets. The p-values for E were calculated using the F-distribution where the degrees of freedom, which depend on the number of exons, were determined by fitting rational polynomials to the expected sum-of-squares in a numerical simulation.

Significance Index and Event Type
We developed a "significance index" calculated as the negative log 10 (p-value) for alternative splicing plus two times the maximum absolute value of the E term, for each gene. Using this significance index, one can order the entire set of genes according to evidence for alternative splicing.
For the genes with evidence of alternative splicing, we define events of four types: Exon gained early, Exon lost early, Exon gained late, Exon lost late, in comparison to the control condition. For each interior exon, we interrogate the junctions between it and its preceding (J1-1st) or its following exon (J1-2nd), and the junction joining the preceding to the following exon (J2). In some cases, the J2 probe was not available. In others, Research. on September 15, 2017. © 2010 American Association for Cancer cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Author Manuscript Published OnlineFirst on September 3, 2010; DOI: 10.1158/0008-5472.CAN-  Author manuscripts have been peer reviewed and accepted for publication but  [E(early,J1-2nd) -E(c, J1-2nd)] < 0. We also required that the magnitude of one of the differences be greater than 2-fold. Not all of the significant genes contained one of these 4 types of events. Some had multiple events, some had both gain and loss of different exons.

High-Throughput GoMiner (HTGM)
HTGM leverages the Gene Ontology (GO) to identify "biological processes" represented in a list of genes. High-Throughput GoMiner (HTGM) (33) was used here, is an enhancement of GoMiner that efficiently processes an arbitrary number of such gene lists. The gene lists obtained from AnovaSVD were sorted in decreasing order with respect to the significance index. We submitted the top 998 genes for HTGM analysis.
The HTGM parameters are listed in Supplemental Table 2.
A GO category is considered to be enriched if the number of changed genes that HTGM assigned to it is greater than the number expected by chance. The enrichment of a category is considered to be statistically significant if its false discovery rate (FDR) is less than or equal to a given threshold (typically 0.10). See (33) for details.

Genesis Clustering Program
Research. Clustered image maps (CIMs) were produced in our studies by the Genesis program (33).
We chose the Euclidean distance metric and average linkage for hierarchical clustering.
Large generic categories were removed from all CIMs to facilitate visualization.

Genome-wide analysis of splicing alterations induced by Top1 poisoning
The ExonHit array allows the analysis of 138,636 splice events among 20,649 genes. The probes are designed to mostly recognize two kind of splicing events: exon skipping and novel exon ( To study the impact of Top1 poisoning on splicing, we purified total RNA from human colon carcinoma HCT116 cells treated with 10 μM CPT for 1, 2, 4, 15 and 20 hours ( Fig. 2A), and performed ExonHit array analysis for each sample. Controls samples were analyzed at 4 and 20 h following DMSO treatment (0.1%; the solvent used to dissolve CPT).
The data were analyzed using a new ExonSVD model (see Materials and Methods) (all the data are in GEO; accession number "GSE23677"; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE23677). Figure 2B shows the  Table 3). The type of alternative splicing event could be determined for 50% of the genes (434 genes) and corresponded to novel exon or exon skipping ( To validate the ExonHit genome-wide results, we took 12 genes which ranked near the top of the list of the 998 genes, according to significance index (Supplemental Table 4) and performed RT-PCR with gene-specific primers. The RT-PCR results were in agreement with the ExonHit results for more than 90% of the splice alterations analyzed (see Supplemental Table 4). showed a decrease of the long transcript (containing exon 5) and the appearance of the short transcript (without exon 5) following CPT treatment. Exon gain was also validated in response to CPT for the caspase-2 gene (Supplemental Fig.1).

Splicing alterations are enriched in genes coding for splicing factors
Next, we tested whether CPT affected the alternative splicing of specific gene families. The 998 differentially spliced genes were analyzed by GoMiner software (http://discover.nci.nih.gov/gominer/index.jsp). The categories of genes that tended to be preferentially affected are listed in Figure 4 and Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Copyright © 2010 American Association for Cancer Research 13 ZRANB2, BAT1 and SF1 genes were further examined (Supplemental Table 4 and Fig.   5). Kinetic experiments were performed for the RBM8A and SF1 genes not only in HCT116 cells but also in human breast carcinoma MCF7 cells treated with CPT. By RT-PCR the effect on splicing for RBM8A, which is the skipping of exon 3, was detected early, beginning 1 h after CPT treatment in both cell lines (Fig. 5A). Splicing of SF1, which corresponds to the skipping of exon 4, was detectable later, at 15 h CPT treatment (Fig. 5B). Together these results demonstrate preferential splicing alterations in the genes encoding splicing factors by Top1 poisoning.

Pol II hyperphosphorylation is associated with Top1-induced splicing alterations
To determine the specificity of CPT in inducing altered splicing, other anticancer agents that do not target Top1 were tested. Neither cisplatin, a DNA alkylating drug, nor vinblastine, a mitotic spindle poison affected the splicing of RBM8A or SF1 (Fig. 6A).
To test whether the effect of CPT was related to Top1cc or due to Top1 depletion by its sequestering in Top1cc, we down-regulated Top1 using an siRNA (Fig. 6B, right panel).
Down-regulation of Top1 had no effect on alternative splicing of the RBM8A or SF1 genes (Fig. 6B, left panel), indicating that the generation of Top1cc rather than Top1 inactivation is necessary to induce splicing.
These results suggest a relationship between CPT-induced Pol II hyperphosphorylation and splicing alterations.

Discussion
This genome-wide analysis study shows that Top1cc alters the splicing of a large to identify the appearance of new splice events by the detection of change (mostly an increase) in E or C and D probes (see Figure 1).  (29). Also, many of the changes observed at 20 hours were already readily detectable at early time (1 hour). Consequently, the changes observed concerning alternative splicing do not mainly reflect the cells going through the process of dying.
Top1 poisoning by CPT and anticancer drugs, abasic sites, base mismatches, oxidized bases, carcinogenic adducts and strand breaks can deplete Top1 activity by sequestering Top1 in the cleavage complexes (7,22,23). However, it is well-established that the anticancer cytotoxic activity of CPT is not due to depletion of Top1 activity but rather to the trapping of Top1cc and subsequent effects of DNA replication and transcription (8,45). Similarly, our Top1 depletion experiments with siRNA show that the splicing effects of CPT are Top1cc-dependent but are probably unrelated to depletion of Top1 catalytic activity. We recently reported the rapid induction of Pol II hyperphosphorylation in response to CPT-induced Top1cc (25). In this study, we confirmed Pol II hyperphosphorylation after CPT treatment. Pretreatment with DRB, which inhibits CDK (25) and suppresses Pol II hyperphosphorylation (see Fig. 6) (25) abrogated the splicing effects of CPT. It is plausible that CPT alters splicing in response to Pol II hyperphosphorylation (see Fig. 7), which has been proposed to affect Pol II Top1cc by CPT can affect splicing by such a "kinetic coupling model" (47). However, it is also possible that hyperphosphorylation of the carboxy terminal domain (CTD) of Pol II affects its interaction with and recruitment of splicing factors as proposed in the "recruitment coupling model" (48,49). In summary, transcription could regulate alternative splicing by modulation of Pol II elongation rates (kinetic coupling) (47)         Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.