Topoisomerase I and II Inhibitors Control Caspase-2 Pre-Messenger RNA Splicing in Human Cells Ste ´ phanie Solier, 1,2 Ame ´ lie Lansiaux, 3 Emmanuelle Logette, 1 Jane Wu, 4 Johann Soret, 5 Jamal Tazi, 5 Christian Bailly, 3 Lydie Desoche, 1 Eric Solary, 1 and Laurent Corcos 1 1 Inserm U517, Faculty of Medicine, Dijon, France; 2 Service d’ He ´ matologie Clinique, CHRU le Bocage, Dijon, France; 3 Inserm U524, Institut de Recherches sur le Cancer de Lille, Lille Cedex, France; 4 Department of Pediatrics, Washington University School of Medicine, McDonnell Pediatric Research Building Rm 3107, St. Louis, MO; and 5 Metazoan Messenger RNAs Metabolism, IGM, UMR 5535 CNRS, Montpellier, France Abstract We have recently shown that the topoisomerase II inhibitor, etoposide (VP16), could trigger caspase-2 pre-mRNA splicing in human leukemic cell lines. This leads to increased inclusion of exon 9, which is specifically inserted into the short caspase-2S isoform mRNA and absent from the long caspase-2L isoform mRNA. One of the consequences of this alternative splicing is a decrease in the total amount of the mature form of caspase-2L mRNA and protein. In this study, we analyzed the effects of several representative molecules of various classes of cytotoxic agents on caspase-2 pre-mRNA splicing in both U937 leukemic cells and in HeLa cervix carcinoma cells. Very strikingly, both topoisomerase I (camptothecin and homocamptothecin derivatives) and II (VP16, amsacrine, doxorubicin, mitoxantrone) inhibitors induced exon 9 inclusion. DNA intercalating glycosyl indolocarbazole derivatives as well as DNA alkylating agents, such as cisplatin and melphalan, antimetabolites like 5-fluorouracil, and mitotic spindle poisons like vinblastine had no effect. Therefore, both classes of DNA topoisomerases can control pre-mRNA splicing of the caspase-2 transcript. In addition, the splicing reaction brought about by camptothecin was hampered in human CEM/C2 and in murine P388-45R leukemic deficient in topoisomerase I activity. Conversely, VP16 did not trigger caspase-2 alternative splicing in human HL60/MX2 leukemic cells harboring a mutant topoisomerase II. Minigene transfection analysis revealed that topoisomerase inhibitors did not change the splicing profile when cis -acting elements in intron-9, reported to control exon 9 inclusion independently of drug treatment, were removed. Rather, our experiments suggest that exon 9 inclusion induced by topoisomerase inhibitors reflects the activity exerted by topoisomerase I or II on proteins that control splicing reactions, or their direct involvement in pre-mRNA splicing. Introduction Recent estimates suggest that about 40 – 60% of pre-mRNAs are subject to alternative splicing, which accounts for proteome diversity under various situations (1, 2). Pre-mRNA splicing requires a multiprotein complex referred to as the spliceosome, which has recently been purified and shown to contain more than 150 polypeptides (3). The activity of splicing factors is controlled at various levels, including subcellular localization and phosphorylation at serine and threonine residues. For example, the activity of the highly conserved Serine-arginine- rich (SR) proteins can be modulated by phosphorylation of serine residues. SR proteins include one or two RNA recognition motifs (RRMs) and a COOH-terminal arginine- serine repeat of varying length (RS domain; for reviews, see Refs. 4 and 5). The RNA recognition motifs mediate recognition of specific RNA determinants known as exonic splicing elements (6, 7), whereas the RS domain is responsible for specific protein-protein interactions (8 – 10). SR proteins that are bound to exonic splicing elements affect splicing by directly recruiting the splicing machinery through their RS domain and/or by antagonizing the action of nearby silencer elements (7, 10). Topoisomerase I has been shown to regulate this activity by phosphorylation of specific serine residues located within the RS domain (11), leading to reprogramming of pre-mRNA splicing of several genes such as CD44 and Bcl- X (12). 6 However, the large implication of the kinase activity of topoisomerases as a key determinant for gene regulation remains to be determined. The expression of numerous genes involved in apoptosis regulation is modulated by alternative splicing (13). These genes are transcribed as diverse mRNA species, which encode proteins with sometimes opposite functions. One example is the CASP-2 gene that codes for a proteolytic enzyme involved in cell dismantling in response to various stimuli such as cytotoxic drugs (14, 15). Alternative splicing of this pre-mRNA generates two major mRNA species (16) including a long caspase-2L isoform that encodes a pro-apoptotic protein and a short Received 7/18/03; revised 11/1/03; accepted 11/14/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Grant support: This work was supported by the INSERM, the Conseil Re ´gional de Bourgogne, the Ligue Nationale Franc ¸aise Contre le Cancer (E. Solary and C. Bailly). S. Solier was financed by the University Ho ˆpital du Bocage (Dijon). E. Logette was recipient of a fellowship from the French Ministry of Education and Research. Requests for reprints: Laurent Corcos, Inserm U517, Faculty of Medicine, 7 Boulevard Jeanne d’Arc, 21000 Dijon, France. Phone: 33-3-80-39-34-40; Fax: 33-3-80-39-34-34. E-mail: Laurent.corcos@u-bourgogne.fr Copyright D 2004 American Association for Cancer Research. 6 J. Soret, unpublished results. Vol. 2, 53 – 61, January 2004 Molecular Cancer Research 53