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,1
* Department of Biochemistry, Virginia Commonwealth University, Richmond, Virginia, USA; and
Research and Development, Hunter Holmes McGuire Veterans Administration Medical Center, Richmond, Virginia, USA
1Correspondence: Department of Biochemistry, Rm. 2–016, Sanger Hall, Virginia Commonwealth University, 1101 East Marshall St., P.O. Box 980614, Richmond, VA 23298-0614, USA. E-mail: cechalfant{at}vcu.edu
SPECIFIC AIMS
Two splice variants are derived from the BCL-x gene, proapoptotic Bcl-x(s) and antiapoptotic Bcl-x(L), via alternative 5' splice site selection. In previous studies, our laboratory identified an RNA cis-element within exon 2 of Bcl-x pre-mRNA that is ceramide-responsive termed ceramide-responsive RNA cis-element 1 (CRCE 1). In this study, our goals were to identify the RNA trans-acting factor that interacted with CRCE 1 and explore the role of this factor in regulating the 5' splice site selection of Bcl-x pre-mRNA. We demonstrate that SAP155 is the RNA trans-acting factor that binds to CRCE 1, functions to regulate the alternative 5' splice site selection of Bcl-x pre-mRNA and is required for ceramide to induce the activation of the Bcl-x(s) 5' splice site. Furthermore, we demonstrate that activation of the Bcl-x(s) 5' splice site can increase the effectiveness of chemotherapeutic drug treatment, thus establishing a role for the alternative splicing mechanism of Bcl-x in chemotherapeutic sensitivity.
PRINCIPAL FINDINGS
1. Identification of RNA trans-acting factors that bind CRCE 1
Previously, our laboratory reported that CRCE 1 was located 277–295 bp upstream of intron 2, within exon 2 of Bcl-x pre-mRNA. To identify the possible RNA trans-acting factors that bind to CRCE 1, in vitro binding assays were performed using a biotinylated oligonucleotide specific for CRCE 1 in the presence of biomagnetic beads covalently linked to streptavidin. The high affinity of biotin for streptavidin allowed for the collection of the CRCE 1:protein complex by magnetic pull-down followed by SDS-PAGE. Specific complexes to CRCE 1 were identified as novel proteins (bands) visualized by Coomassie blue staining as compared with a biotinylated control (scrambled) RNA oligo and magnetic beads. Gel bands containing proteins that specifically bound to CRCE 1 were then subjected to ingel tryptic digestion and mass spectrometric peptide sequencing for protein identification. These peptide sequences were then used to determine a "best fit" to a theoretical in silico tryptic digest and MS/MS sequencing of the protein database. In this manner, SAP155 was identified as possible RNA trans-acting factor that binds CRCE 1.
2. SAP155 regulates the 5' splice site selection of Bcl-x pre-mRNA
To determine the RNA splicing factor bound to CRCE 1 that played a role in regulating the alternative 5' splice site selection of Bcl-x pre-mRNA, RNA interference (RNAi) technology was used to down-regulate these splicing factors in A549 cells. A "pool" of siRNA targeting SAP155 resulted in an 
85% knockdown of SAP155 as determined by Western blot analysis. Down-regulation of SAP155 induced an increase in Bcl-x(s) splice site selection at the expense of Bcl-x(L), thereby inducing a decrease in the Bcl-x(L)/Bcl-x(s) ratio from 7.98 ± 0.69 to 2.62 ± 0.12, P < 0.005. The effects on mRNA levels translated to the protein concentration as down-regulation of SAP155 also decreased the immunoreactive protein levels of Bcl-x(L) by 40% with a concomitant 2.3-fold increase in the immunoreactive protein levels of Bcl-x(s). Three approaches were undertaken to demonstrate the specificity of SAP155 siRNA and to control for off-target effects. First, we examined individual siRNAs (SAP155–1, SAP155–2, SAP155–3, and SAP155–4) against SAP155. Each individual siRNA to SAP155 induced an increase in the Bcl-x(s) splice variant with a concomitant decrease in the Bcl-x(L) splice variant demonstrating that the effect on Bcl-x pre-mRNA processing is specific for the down-regulation of SAP155 and not due to off-target effects of the siRNA. Second, because off-target activity is reduced in a concentration-dependent manner, we examined the effective concentration of the SAP-155 siRNA. Low concentrations of SAP155 siRNA (10 and 25 nM) also induced activation of the Bcl-x(s) 5' splice site in a similar manner as the treatment concentration (200 nM) of SAP155 siRNA. Third, siRNA targeted for SAP155 had no effect on the expression of control splicing factors.
The effect of down-regulation of SAP155 on Bcl-x pre-mRNA processing was not due to a generalized effect on the RNA splicing machinery, as the alternative splicing of caspase-9 and Bax were unaffected. Furthermore, down-regulation of the RNA trans-acting factor associated with SAP155 for constitutive splicing activity, U2AF65 (4), had no effect on the 5' splice site selection of Bcl-x. Finally, down-regulation of SAP145, 130, and 49, which together with SAP155 are the spliceosomal protein constituents of SF3b, an essential complex that associates with U2 snRNP for constitutive splicing activity, also had no effect on the alternative splicing pattern of Bcl-x. Therefore, these data demonstrate that SAP155 specifically regulates the alternative 5' splice site selection of Bcl-x pre-mRNA, and this effect on the Bcl-x(L)/Bcl-x(s) ratio is not due to an effect on the overall activity of pre-mRNA processing.
3. SAP155 specifically interacts with CRCE 1
The specificity of the SAP155:CRCE 1 complex was further demonstrated by EMSAs and PAGE-SDS analysis. In the first study, the specific binding of SAP155 to CRCE 1 was shown by including an anti-SAP155 antibody (Ab) in EMSAs. As previously reported by our laboratory, EMSAs using A549 nuclear extract and a fluoroscein-tagged oligonucleotide specific for CRCE 1 induced a reduction in the migration of the oligonucleotide as a result of RNA-protein(s) complex formation. To determine whetherSAP155 was associated to CRCE 1, an anti-SAP155 Ab (rabbit) or a rabbit IgG (control) was incorporated in the EMSA. The addition of the anti-SAP155 Ab produced a specific CRCE 1:protein complex of reduced migration, whereas, the addition of IgG had no effect on the mobility of the specific CRCE 1:protein complex.
In a second study, RNAi technology was coupled to SDS-PAGE analysis. These assays were performed using a fluoroscein-tagged oligonucleotide specific for CRCE 1, as well as nuclear extract from A549 cells transfected with either SAP155 or control siRNA. The use of RNAi technology to generate SAP155-deficient nuclear extracts resulted in the loss of a specific 155 kDa protein complex to CRCE 1. Taken together, these data, coupled to the results of mass spectrometric analysis, indicate that SAP155 associates specifically with CRCE 1.
4. SAP155 is a required RNA trans-acting factor for the activation of the Bcl-x(s) 5' splice site in response to ceramide
Exogenous ceramide was previously shown to activate the Bcl-x(s) 5' splice site requiring increased CRCE 1:protein complex formation. Based on these data, we hypothesize that down-regulation of the ceramide-responsive RNA trans-acting factor (CRTF) will inhibit the ability of ceramide to modulate the alternative splicing pattern of Bcl-x pre-mRNA. Furthermore, we hypothesize that if ceramide is modulating the 5' splice site selection of Bcl-x pre-mRNA via a different RNA trans-acting factor, then an additive/synergistic effect of SAP155 siRNA and ceramide treatment on the Bcl-x(L)/Bcl-x(s) ratio will be observed. To examine this hypothesis, A549 cells subjected to SAP155 or control siRNA were treated with 20 µM D-erythro-C6 ceramide for 24 h (maximal response time). Analysis of the alternative splice variants of Bcl-x revealed that treatment in the absence of SAP155 siRNA activated the Bcl-x(s) 5' splice site, as we have previously reported, thereby changing the Bcl-x(L/s) ratio from 12.61 ± 0.32 to 4.97 ± 0.01. A549 cells treated with only SAP155 siRNA also induced the Bcl-x(s) 5' splice site selection to a similar extent as ceramide (24 h), thereby changing the Bcl-x(L/s) ratio from 12.61 ± 0.32 to 3.92 ± 0.015. Ceramide treatment of cells subjected to SAP155 siRNA did not significantly induce the activation of the Bcl-x(s) 5' splice site beyond the capacity of SAP155 down-regulation alone (Fig. 1
A). The inability of ceramide to induce the processing of the proapoptotic alternative splice variant was specific to the Bcl-x alternative splicing mechanism, as the ceramide-induced processing of caspase-9a was unaffected by the down-regulation of SAP155 (Fig. 1B
). Therefore, down-regulation of SAP155 inhibited the ability of exogenous ceramide treatment to further activate the Bcl-x(s) 5' splice site demonstrating that SAP155 is required for activation/preferential use of the Bcl-x(s) 5' splice site in response to ceramide.
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5. Down-regulation of SAP155 sensitized cells to undergo apoptosis in response to daunorubicin
Previous studies have demonstrated that the activation of the Bcl-x(s) 5' splice site at the expense of Bcl-x(L) sensitized cells to chemotherapy and ultimately led to apoptosis. Our laboratory also reported that treatment of A549 cells with ceramide induced a similar effect on Bcl-x pre-mRNA processing, and this treatment sensitized cells to chemotherapeutic drug treatment (e.g., daunorubicin). Therefore, we examined for the ability of the down-regulation of SAP155 (demonstrated in these studies to activate the Bcl-x(s) 5' splice site) to sensitize A549 cells to the chemotherapeutic agent, daunorubicin. A549 cells transfected with SAP155 siRNA or control (scrambled) siRNA were treated with various doses of daunorubicin and assessed at several time points for cell viability and growth. Transfection of A549 cells with SAP155 siRNA sensitized cells to undergo apoptosis after treatment with a low dose of the chemotherapeutic agent, daunorubicin (lowered the IC50 from 0.102 to 0.033 µM; P<0.01). Treatment of A549 cells with D-erythro-C6 ceramide similarly sensitized cells to undergo apoptosis in response to daunorubicin (also lowered the IC50 from 0.102 to 0.012 µM; P<0.01). Interestingly, the combined transfection of A549 cells with SAP155 siRNA and treatment with D-erythro-C6 ceramide did not have an additive/synergistic effect on the sensitization of cells to undergo apoptosis in response to drug treatment. Furthermore, this sensitization to daunorubicin occurred after only short term (<48 h) down-regulation of SAP155. Thus, using RNAi technology to down-regulate the RNA splicing factor, SAP155, induces pre-mRNA processing of the proapoptotic Bcl-x(s) and correlates with the sensitization of A549 cells to daunorubicin and loss of viability in A549 cells in accordance with previous reports. However, combined treatments of ceramide and SAP155 siRNA (long term) did not intensify the loss of viability in A549 cells implicating the alternative splicing mechanism of Bcl-x as a major mechanism of ceramide-induced cell death.
CONCLUSIONS AND SIGNIFICANCE
In this study, we identified SAP155 as an RNA trans-acting factor that binds to CRCE 1 and functions to regulate the alternative 5' splice site selection of Bcl-x pre-mRNA. Ceramide was unable to activate/induce preferential use of the Bcl-x(s) 5' splice site beyond the capacity of SAP155 siRNA, suggesting that SAP155 is required for the ability of ceramide to induce the activation of the Bcl-x(s) 5' splice site. These findings are important for several reasons. First, they provide further insight to the mechanistic understanding of the RNA splicing factors involved in the regulation of the alternative splicing of Bcl-x in response to ceramide. Second, a novel function for SAP155 in the regulation of alternative 5' splice site selection has been established. Finally, a direct link between a signal transduction pathway mediating the 5' alternative splice site selection of Bcl-x pre-mRNA processing and sensitivity to chemotherapeutic agents has been substantiated.
The demonstration that SAP155 regulates the alternative 5' splice site selection of Bcl-x pre-mRNA is very novel as previous studies by Reed and coworkers have suggested that SAP155 is an essential splicing factor necessary for specific protein interactions for spliceosomal assembly. In the present study, down-regulation of SAP155 only affected the 5' splice site selection of Bcl-x pre-mRNA, and had no affect on the splice variant patterns of other apoptotic pre-mRNAs such as caspase 9 and Bax. Furthermore, down-regulation of human U2AF, the RNA splicing factor shown to interact with SAP155 in the spliceosome, and also required for yeast viability and growth, had neither effect on the alternative 5' splice site selection of Bcl-x pre-mRNA nor cell viability. Therefore, this study demonstrates that SAP155 can have dual roles in mammalian cells, as both a regulator of alternative splicing (e.g., alternative 5' splice site selection), and as a constitutive RNA splicing factor. The demonstration of an RNA trans-acting factor having a dual role in RNA splicing is not without precedent as SR proteins are well-characterized as having roles in constitutive and alternative splicing. Furthermore, this study also implicates SAP155 as a novel downstream target of ceramide that may be involved in a ceramide-mediated signal transduction pathway for the induction of apoptosis.
The identification of this new role for SAP155 is quite interesting as our laboratory previously demonstrated that the 5' splice site selection of Bcl-x pre-mRNA was regulated by a PP1-dependent mechanism. Recently, Bollen and coworkers have demonstrated that PP1 is associated with SAP155 via the nuclear inhibitor of protein phosphatase-1 (NIPP1). In the same study, they further demonstrated that this interaction is controlled by multisite phosphorylation, thus, PP1 is in proximity to SAP155 and a regulator of its phospho-state. These data are consistent with our findings that Bcl-x processing is mediated via the activation of PP1 by de novo ceramide. Taken together, these studies implicate a possible role of PP1 in the 5' splice site selection of Bcl-x pre-mRNA via regulation of the phospho-status of SAP155, and implicate SAP155 as a novel target molecule of ceramide and substrate of PP1 for the induction of cell death (Fig. 2
).
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Physiological significance of the Bcl-x(L)/Bcl-x(s) ratio has been documented by many reports in the literature demonstrating that the fate of the cell can be determined by the proportion of antiapoptotic Bcl-x(L) to proapoptotic Bcl-x(s). Thus, SAP155 is a novel factor in the signal transduction pathway that regulates the alternative splicing mechanism of Bcl-x and sensitivity of lung adenocarcinoma cells to apoptosis. This mechanism of regulation has direct relevance to drug resistance and chemotherapeutic sensitivity since specific regulation of alternative splicing of Bcl-x is linked to sensitization of cells to chemotherapy, giving rise to a new target for anticancer therapies.
FOOTNOTES
SYSTEMS NOTE: This article was paginated as 3 pages ending on 1690 per DR(from SS)
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5021fje
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