Previous studies have demonstrated that several splice variants are derived from both the caspase 9 and Bcl-x genes in which the Bcl-x splice variant, Bcl-x(L) and the caspase 9 splice variant, caspase 9b, inhibit apoptosis in contrast to the pro-apoptotic splice variants, Bcl-x(s) and caspase 9. In a recent study, we showed that ceramide induces the dephosphorylation of SR proteins, a family of protein factors that regulate alternative splicing. In this study, the regulation of the alternative processing of pre-mRNA of both caspase 9 and Bcl-x(L) was examined in response to ceramide. Treatment of A549 lung adenocarcinoma cells with cellpermeable ceramide, D-e-C 6 ceramide, down-regulated the levels of Bcl-x(L) and caspase 9b mRNA and immunoreactive protein with a concomitant increase in the mRNA and immunoreactive protein levels of Bcl-x(s) and caspase 9 in a dose-and time-dependent manner. Pretreatment with calyculin A (5 nM), an inhibitor of protein phosphatase-1 (PP1) and protein phosphatase 2A (PP2A) blocked ceramide-induced alternative splicing in contrast to okadaic acid (10 nM), a specific inhibitor of PP2A at this concentrations in cells, demonstrating a PP1-mediated mechanism. A role for endogenous ceramide in regulating the alternative splicing of caspase 9 and Bcl-x was demonstrated using the chemotherapeutic agent, gemcitabine. Treatment of A549 cells with gemcitabine (1 M) increased ceramide levels 3-fold via the de novo sphingolipid pathway as determined by pulse labeling experiments and inhibition studies with myriocin (50 nM), a specific inhibitor of serine palmitoyltransferase (the first step in de novo synthesis of ceramide). Treatment of A549 cells with gemcitabine downregulated the levels of Bcl-x(L) and caspase 9b mRNA with a concomitant increase in the mRNA levels of Bclx(s) and caspase 9. Again, inhibitors of ceramide synthesis blocked this effect. We also demonstrate that the change in the alternative splicing of caspase 9 and Bcl-x occurred prior to apoptosis following treatment with gemcitabine. Furthermore, doses of D-e-C 6 ceramide that induce the alternative splicing of both caspase 9 and Bcl-x-sensitized A549 cells to daunorubicin. These data demonstrate a role for protein phosphatases 1 (PP1) and endogenous ceramide generated via the de novo pathway in regulating this mechanism. This is the first report on the dynamic regulation of RNA splicing of members of the Bcl-2 and caspase families in response to regulators of apoptosis.
Two splice variants derived from the BCL-x gene, proapoptotic Bcl-x(s) and anti-apoptotic Bcl-x(L), are produced via alternative 5 splice site selection within exon 2 of Bcl-x pre-mRNA. In previous studies, our laboratory demonstrated that ceramide regulated this 5 splice site selection, inducing the production of Bcl-x(s) mRNA with a concomitant decrease in Bcl-x(L) correlating with sensitization to chemotherapy (Chalfant, C. E., Rathman, K., Pinkerman, R. L., Wood, R. E., Obeid, L. M., Ogretmen, B., and Hannun, Y. A. (2002) J. Biol. Chem. 277, 12587-12595). We have now identified several possible RNA cis-elements within exon 2 of Bcl-x pre-mRNA by sequence analysis. To study the possible roles of these RNA cis-elements in regulating the alternative 5 splice site selection of Bcl-x pre-mRNA, we developed a BCL-x minigene construct which conferred the same ratio of Bcl-x(L)/Bcl-x(s) mRNA as the endogenous Bcl-x and was responsive to ceramide treatment. Mutagenesis of either a purine-rich splicing enhancer or a pyrimidine tract element within exon 2 induced a change in the ratio of Bcl-x(L)/Bcl-x(s) mRNA from 7 to 1 and 0.23, thereby diminishing the selection of the Bcl-x(L) 5 splice site with a concomitant increase in Bcl-x(s) 5 splice site selection. Furthermore, mutagenesis of these cis-elements abolished the ability of ceramide to affect the 5 splice site selection. In vitro binding assays coupled with competitor studies demonstrated specific binding of RNA trans-activating proteins to these regions. SDS-PAGE analysis of cross-linked RNA transactivating factors with these RNA cis-elements revealed the binding of 215-, 120-, and 30-kDa proteins to the purine-rich element and 120-and 76-kDa proteins to the pyrimidine tract element. In addition, exogenous treatment of A549 cells with ceramide increased the formation of protein complexes with these RNA cis-elements. Therefore, we have identified two ceramide-responsive RNA cis-elements within exon 2 of Bcl-x pre-mRNA, and this is the first report of an RNA cis-element responsive to a bioactive lipid.Ceramide is an important regulator of various stress responses and growth mechanisms, and the formation of ceramide from the hydrolysis of sphingomyelin or from de novo pathways has been observed in response to agonists such as tumor necrosis factor-␣, ␥-interferon, 1␣,25-dihydroxyvitamin D 3 , interleukin-1, ultraviolet light, heat, chemotherapeutic agents, fatty-acid synthase antigen, and nerve growth factor (1-7). Also, the addition of exogenous ceramide or the enhancement of cellular levels of ceramide induces cell differentiation, cell cycle arrest, apoptosis, or cell senescence in various cell types (8 -10).The prominent role of ceramide as a regulator of cellular mechanisms necessitated the identification of target molecules. A family of ceramide-regulated enzymes has been identified, ceramide-activated protein phosphatases, which include the serine/threonine-specific protein phosphatases PP1 1 and PP2A (11-14). Interestingly, SR proteins, a family of arginine/...
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 thirteen 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 four 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 IC50 of non-small cell lung cancer (NSCLC) cells to daunorubicin, cisplatinum, and paclitaxel. In contrast, downregulation of caspase 9b induced a decrease in the the IC50 of these chemotherapeutic drugs. Lastly, these studies demonstrated 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 demonstrate that the alternative splicing of caspase 9 is an important molecular mechanism with therapeutic relevance to NSCLCs.
Supplementary Figures S1-S7 from 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
Supplementary Figures S1-S7 from 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
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.