title: SRSF1 regulates the alternative splicing of caspase 9 via a novel intronic splicing enhancer affecting the chemotherapeutic sensitivity of non-small cell lung cancer cells.
abstract: Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology with the alternative pre-mRNA processing of caspase 9 as one example. In this study, we delve into the underlying molecular mechanisms that regulate the alternative splicing of caspase 9. Specifically, the pre-mRNA sequence of caspase 9 was analyzed for RNA cis-elements known to interact with SRSF1, a required enhancer for caspase 9 RNA splicing. This analysis revealed 13 possible RNA cis-elements for interaction with SRSF1 with mutagenesis of these RNA cis-elements identifying a strong intronic splicing enhancer located in intron 6 (C9-I6/ISE). SRSF1 specifically interacted with this sequence, which was required for SRSF1 to act as a splicing enhancer of the inclusion of the 4 exon cassette. To further determine the biological importance of this mechanism, we employed RNA oligonucleotides to redirect caspase 9 pre-mRNA splicing in favor of caspase 9b expression, which resulted in an increase in the IC(50) of non-small cell lung cancer (NSCLC) cells to daunorubicin, cisplatinum, and paclitaxel. In contrast, downregulation of caspase 9b induced a decrease in the IC(50) of these chemotherapeutic drugs. Finally, these studies showed that caspase 9 RNA splicing was a major mechanism for the synergistic effects of combination therapy with daunorubicin and erlotinib. Overall, we have identified a novel intronic splicing enhancer that regulates caspase 9 RNA splicing and specifically interacts with SRSF1. Furthermore, we showed that the alternative splicing of caspase 9 is an important molecular mechanism with therapeutic relevance to NSCLCs.
authors.affiliation: Department of Biochemistry, Virginia Commonwealth University-School of Medicine, Richmond, VA 23298-0614, USA.
authors.forename: Jacqueline C
authors.initials: JC
authors.lastname: Shultz
authors.forename: Rachel W
authors.initials: RW
authors.lastname: Goehe
authors.forename: Charuta S
authors.initials: CS
authors.lastname: Murudkar
authors.forename: Dayanjan S
authors.initials: DS
authors.lastname: Wijesinghe
authors.forename: Eric K
authors.initials: EK
authors.lastname: Mayton
authors.forename: Autumn
authors.initials: A
authors.lastname: Massiello
authors.forename: Amy J
authors.initials: AJ
authors.lastname: Hawkins
authors.forename: Prabhat
authors.initials: P
authors.lastname: Mukerjee
authors.forename: Ryan L
authors.initials: RL
authors.lastname: Pinkerman
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Park
authors.forename: Charles E
authors.initials: CE
authors.lastname: Chalfant
mesh_terms: D017398:Alternative Splicing; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000235:genetics; D053453:Caspase 9 / Q000235:genetics*; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000627:therapeutic use; D003630:Daunorubicin / Q000627:therapeutic use*; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D004357:Drug Synergism; D004742:Enhancer Elements, Genetic; D000069347:Erlotinib Hydrochloride; D006367:HeLa Cells; D006801:Humans; D007438:Introns / Q000235:genetics; D008171:Lung Diseases / Q000188:drug therapy* / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D017239:Paclitaxel / Q000627:therapeutic use; D011799:Quinazolines / Q000627:therapeutic use*; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D022821:RNA Splice Sites / Q000235:genetics; D016372:RNA, Antisense / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors
chemical_list: D009687:Nuclear Proteins; D011799:Quinazolines; D012322:RNA Precursors; D022821:RNA Splice Sites; D016372:RNA, Antisense; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D000069347:Erlotinib Hydrochloride; D053453:Caspase 9; D017239:Paclitaxel; D002945:Cisplatin; D003630:Daunorubicin