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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
ABSTRACT
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 a ceramide responsive termed CRCE 1. In this study, mass spectrometric analysis identified the splicing factor SAP155, as an RNA trans-acting factor binding to the purine-rich CRCE 1. The interaction of SAP155 with CRCE 1 was confirmed by the addition of an anti-SAP155 antibody (Ab) to EMSA decreasing the mobility of a protein:CRCE 1 complex (SuperShift). Furthermore, the down-regulation of SAP155 in A549 cells by RNA interference (RNAi) technology resulted in the loss of a 155 kDa protein complexed with CRCE 1. Moreover, this down-regulation of SAP155 induced an increase in the Bcl-x(s) with a concomitant decrease in the Bcl-x(L) splice variants and immunoreactive protein levels, thereby decreasing the Bcl-x(L)/Bcl-x(s) ratio. Specific down-regulation of SAP155 also inhibited the ability of exogenous ceramide treatment to further induce the activation of the Bcl-x(s) 5' splice site. Additionally, the specific down-regulation of SAP155 sensitized cells to undergo apoptosis in response to daunorubicin in a manner similar to ceramide. Therefore, we have identified SAP155 as an 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 have demonstrated 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.—Massiello, A., Roesser, J. R., Chalfant, C. E. SAP155 binds to ceramide-responsive RNA cis-element 1 and regulates the alternative 5' splice site selection of Bcl-x pre-mRNA.
Key Words: Bcl-x • alternative splicing • A549 cells • daunorubicin • chemotherapy
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-
, 
-IFN, 1,-25-dihydroxyvitamin D3, interleukin-1, ultraviolet light, heat, chemotherapeutic agents, FAS antigen, and nerve growth factor (1
2
3
4
5
6
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
9
10)
.
The prominent role of ceramide as a regulator of cellular mechanisms necessitated the identification of direct targets. A family of ceramide-regulated enzymes was identified and termed ceramide-activated protein phosphatases (CAPP), which include the serine-threonine protein phosphatases PP1 and PP2A. Therefore, CAPP activation was a possible mechanism via which ceramide regulated apoptotic signaling. To this end, our laboratory described a pathway linking the generation of ceramide and the activation of PP1 to the regulation of the alternative 5' splice site selection of Bcl-x pre-mRNA (11)
. The BCL-x gene, via alternative 5' splice site selection, is processed to either the proapoptotic Bcl-x(s) upstream 5' splice site selection or the antiapoptotic Bcl-x(L) downstream 5' splice site selection. Ceramide treatment resulted in a decrease in Bcl-x(L) mRNA with a concomitant increase in Bcl-x(s) mRNA in A549 cells. This effect required the generation of endogenous ceramide through the de novo pathway, and, more importantly, inhibitors of protein phosphatase-1 abolished the ability of ceramide to affect the alternative splicing of Bcl-x.
The mechanism of alternative 5' splice site selection of Bcl-x pre-mRNA has emerged as a potential target for anticancer therapeutics as demonstrated in studies by Taylor et al. (12)
. They showed that Bcl-x alternative splicing was specifically modulated using an antisense oligonucleotide specific for Bcl-x pre-mRNA at a site surrounding the Bcl-x(L) 5' splice site. Hybridization of this oligonucleotide to Bcl-x pre-mRNA induced an increase in the expression of Bcl-x(s) and a decrease in the expression of Bcl-x(L), resulting in sensitization of the cells to chemotherapeutic agents and eventually inducing apoptosis after long-term exposure (>48 h). Thus, the alternative splicing of Bcl-x is one process by which ceramide, via PP1, mediates the apoptotic signaling pathway and establishes the importance of alternative splicing mechanisms in cell fate.
Recently, our laboratory identified ceramide-responsive RNA cis-element 1 (CRCE 1) located in exon 2 of Bcl-x pre-mRNA 277–295 bp upstream from intron 2 (13)
. Manipulation of CRCE 1 by replacement mutagenesis dramatically reduced the ratio of Bcl-x(L)/Bcl-x(s) from 7 to 1 and also inhibited exogenous ceramide from further decreasing this ratio (13)
. Additionally, these studies demonstrated that ceramide regulated the formation of protein:RNA complex at CRCE 1. The presented study identifies the involvement of the splicing factor SAP155, as binding to CRCE 1 and a required RNA trans-factor for the alternative 5' splice site selection of Bcl-x in response to ceramide.
MATERIALS AND METHODS
Cell Culture
A549 adenocarcinoma cells were grown in 50% RPMI 1640 (Invitrogen) and 50% Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with L-glutamine, 10% (v/v) FBS (Invitrogen), 100 U/ml of penicillin G sodium (Invitrogen), and 100 µg/ml of streptomycin sulfate (Invitrogen). Cells were maintained at <80% confluency under standard incubator conditions (humidified atmosphere, 95% air, 5% CO2, 37°C). For treatments with D-erythro-C6 ceramide (Matreya), A549 cells were plated at 4 x 105 cells/35 mm plate in the same media.
EMSA
The following sequence was used for FITC-tagged RNA oligonucleotide: CRCE 1: r(5'Fl-GAG GGA GGC AGG CGA CGA C). RNA-binding reactions were performed in a final volume of 25 µl containing: 100 ng CRCE 1 fluorescein-oligonucleotide, 50 µg nuclear protein extract, 40 U RNasin, and 11.3 µg tRNAs in buffer composed of 10 mM HEPES, 1 mM DTT, 120 mM KCl, 3 mM MgCl2, and 5% glycerol. The reaction mixtures were incubated at 4°C for 20 min. Samples were loaded on a 5% TBE-polyacrylamide gel (60:1 acrylamide/bis-acrylamide, see Results; 27:1 acrylamide/bis-acrylamide see Supplemental Fig. 1) for electrophoretic separation of RNA-protein(s) complexes for 1.5 h at 115 V at 4°C. The gel was then scanned using Molecular Imager FX (Bio-Rad) with a 488 nm EX (530 nm BP) laser. For supershift reactions, Ab to SAP155 (provided by Dr. Robin Reed, Harvard Medical School, Boston, MA) or IgG (Sigma) was added 20 min after addition of the oligonucleotide with an additional 30 min of binding time at 4°C. Where indicated, the SAP155 Ab was heat-denatured at 57°C for 60 min. Samples were then subjected to electrophoretic separation and analyzed as described above.
SDS-PAGE Analysis
Following the same procedures for the binding reactions described for EMSA (above), samples were boiled in Laemmli buffer for 10 min and then subjected to 7.5% SDS-PAGE for 30 min at 50 V followed by 90 min at 100 V for determination of the molecular weights of the separated RNA-protein(s) complexes. The gel was then scanned using a Molecular Imager FX (Bio-Rad) with a 488 nm EX (530 nm BP) laser.
Bio-Magnetic Bead-Streptavidin Pull-down Assay
A biotinylated oligonucleotide for CRCE 1: r(5'Bi-GAG GGA GGC AGG CGA CGA C; Dharmacon) was incubated under EMSA binding conditions (above) with A549 nuclear extract. After 20 min incubation at 4°C, 10 µl of 10 mg/ml Dynabeads MyOn streptavidin (Dynal Biotech) were added to the reaction assay and incubated with agitation for 30 min at 4°C. After this incubation, the complex was pelleted for 1 min by magnetic pull-down. The pellet was resuspended in 25 µl 1x Laemmli lysis buffer and boiled 10 min for SDS-PAGE analysis.
Mass Spectrometric Analysis
Identification of protein complexes was performed at the UNC Michael Hooker Proteomics Core Facility. Briefly, an automated ingel tryptic digestion was performed on manually excised selected gel bands. The extracts were analyzed by LC-MS/MS, an approach used for complex mixtures where the separation of peptides is done on-line before ESI-MS/MS. Protein identification by LC-MS/MS relies on a "sequence-tag" approach, which is based on tandem mass spectrometric sequencing of a peptide through collision-induced dissociation. Results were obtained by searching the Mascot (Matrix Science; London, England) database (14)
.
Reverse Transcription-Polymerase Chain Reaction Assay
Total RNA from A549 cells was isolated using the RNeasy kit (Qiagen) per manufacturer’s protocol. One microgram of A549 total RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen) and oligo(dT) as the priming agent. After 50 min incubation at 42°C, the reactions were stopped by 70°C heating for 15 min. Template RNA was then removed using RNase H (Invitrogen).
For evaluating the expression of Bcl-x splice variants, an upstream 5' primer to Bcl-x (5'-GAG GCA GGC GAC GAG TTT GAA-3') and a 3' primer (5'-TGG GAG GGT AGA GTG GAT GGT-3'; Integrated DNA Technologies) were used. With the use of these primers, 20% of the reverse transcriptase reaction was amplified for 35 cycles (94°C, 30 s melt; 58°C, 30 s anneal; 72°C, 1 min extension) using Platinum TaqDNA polymerase (Invitrogen). The polymerase chain reaction (PCR) was examined by 1.5% agarose gel electrophoresis. The gel was then stained with SYBR Gold (Invitrogen) and scanned using a Molecular Imager FX (Bio-Rad) with a 488 nm EX (530 nm BP) laser.
Small interfering RNA transfection
Transfection of A549 cells with the SAP155, U2AF65, SAP145, SAP130, or SAP49 SMARTpool or SAP155 SMARTselection designed siRNA reagents (Dharmacon) was performed using oligofectamine (Invitrogen) following the manufacturer’s (Invitrogen) protocol. The SMARTselection duplex RNA targeting sequences used were the following: SAP155–1 sense sequence (5'-GGA AUU AGA UGC UAU GUU CUU-3') and antisense sequence (5'-GAA CAU AGC AUC UAA UUC CUU-3'); SAP155–2 sense sequence (5'-GCA AAC GAG UCA AAC CAU AUU-3') and antisense sequence (5'-UAU GGU UUG ACU CGU UUG CUU-3'); SAP155–3 sense sequence (5'-GAA CCG CUA UUG AUU GAU GUU-3') and antisense sequence (5'-CAU CAA UCA AUA GCG GUU CUU-3'); and SAP155–4 sense sequence (5'-GUA GAA UGU UGC AAU AUU GUU-3') and antisense sequence (5'-CAA UAU UGC AAC AUU CUA CUU-3'). Briefly, A549 cells were plated in regular growth medium at 40–50% confluency in a sixwell tissue culture dish 24 h before transfection. Cells in 1.8 µl Opti-Mem I medium without antibiotics/FBS were transfected with 200 nM (dilution in Opti-Mem I) of the oligonucleotide (in 15 µl of 4 µl Oligofectamine/Opti-MEM I reduced serum medium and incubated for 4 h at standard incubator conditions. After the incubation, 0.5 µl Opti-MEM I reduced serum medium containing three times the normal concentration of antibiotics/FBS was added to the transfected A549 cells without removing the transfection mixture. After 48 h, total RNA isolation or total protein lysates were collected as described for examination by reverse transcription (RT)-PCR or Western blot analysis.
Nuclear Extracts
Nuclear extracts were prepared from A549 lung adenocarcinoma cells according to the method of Dignam et al. (15)
as described previously (13)
. Protein concentrations were determined by a modification of the Bradford method using the Bio-Rad protein assay reagent.
Protein Extraction
Total protein was extracted by direct lysis with Laemmli buffer. Cells were lysed with 0.1 µl of 2x Laemmli buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.04% bromphenol blue, and 250 mM ß-mercaptoethanol) after resuspension in 0.1 µl of ice-cold PBS. Samples were boiled for 10 min and either examined directly by SDS-PAGE or stored at –20°C.
Western Immunoblotting
Total protein lysate (20 µg) was subjected to 7.5, 10, or 15% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad) and blocked in 5% milk, 1x PBS-T (M-PBS-T) for 2 h. The membrane was incubated with anti-SAP155 (Reed), anti--tubulin (Santa Cruz Biotechnology), anti-Bcl-x(L) (Santa Cruz Biotechnology), anti-Bcl-x(s) (Oncogene), or anti-U2AF65 (Zymed) for 2 h in M-PBS-T followed by three washes with PBS-T. The membrane was then incubated with a secondary Ab of horseradish peroxidase-conjugated goat anti-rabbit IgG (anti-SAP155; Pierce), horseradish peroxidase-conjugated anti-mouse IgM (anti--tubulin; Cal-Biochem), horseradish peroxidase-conjugated goat anti-rabbit (anti-Bcl-x; Pierce), or horseradish peroxidase-conjugated goat anti-mouse IgG (anti-U2AF65; Pierce) for 45 min followed by three washes with PBS-T. Immunoblots were developed using Pierce enhanced chemiluminescence (ECL) reagents and Bio-Max film.
MTT Assay
A549 cells (1.0 x 105) were plated into each well of a 24-well plate in a 1 µl vol. After 24 h at standard incubator conditions (humidified atmosphere, 95% air, 5% CO2, 37°C), the cells were transfected with siRNA following the manufacturer’s (Invitrogen) protocol (briefly described above). Cells were treated with the appropriate concentration of daunorubicin (nine hours post-transfection) or ceramide (33 h post-transfection) in a 0.5 µl volume and returned to the incubator. After the appropriate time, 50 µl of MTT solution (5 mg/ml) were added and cells were again incubated under standard conditions for 4 h. Cells were then lysed and solubilized by the addition of 0.5 µl of MTT solvent (0.1 N HCl in anhydrous isopropanol). The plate was read at A595.
RESULTS
Identification of RNA trans-acting factors that interact with 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 (13)
. 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 (data not shown). 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 (14)
. In this manner, several proteins were identified as possible RNA trans-acting factors that bind CRCE 1 (Table 1
). Table 1
also depicts several RNA trans-acting factors suggested in the literature to bind purine-rich sequences, regulate apoptosis, or affect the alternative splicing of Bcl-x pre-mRNA (16
17
18
19
20)
.
|
SAP155 regulates the 5' splice site selection of Bcl-x pre-mRNA
To determine which of the splicing factors listed in Table 1
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 approximate 85% knockdown of SAP155 as determined by Western blot analysis (Fig. 1
A). 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 (Fig. 1B
). 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) (Fig. 1C
). Three approaches were undertaken to demonstrate the specificity of SAP155 siRNA and to control for off-target effects (16)
. 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 SAP155 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 affect on the other splicing factors listed in Table 1
. Down-regulation of the other splicing factors (confirmed by western immunoblotting as >75%) listed in Table 1
by RNAi technology did not induce any change in the 5' splice site selection of Bcl-x pre-mRNA (data not shown).
|
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 (Fig. 1D
). Furthermore, down-regulation of the RNA trans-acting factor associated with SAP155 for constitutive splicing activity, U2AF65 (21)
, had no affect on the 5' splice site selection of Bcl-x (Fig. 1E
). 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 (17)
, also had no effect on the alternative splicing pattern of Bcl-x (Fig. 1F
). 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.
SAP155 specifically interacts with CRCE 1
The specificity of the SAP155:CRCE 1 complex was further demonstrated by electromobility gel shift and PAGE-SDS analysis. In the first study, the specific binding of SAP155 to CRCE 1 was shown by including an anti-SAP155 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 (13
; Supplemental Fig. 1). 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 (for Fig. 2
A, lane 2), whereas the addition of IgG had no effect on the mobility of the specific CRCE 1:protein complex (Fig. 2A
, lane 1). Moreover, the addition of a denatured anti-SAP155 Ab also had no effect on the mobility of the specific CRCE 1:protein complex (see Supplemental Fig. 1).
|
In a second study, RNAi technology was coupled to PAGE-SDS 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 (Fig. 2B
). Taken together, these data, coupled to the results of mass spectrometric analysis, indicate that SAP155 associates specifically with CRCE 1.
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 (11
, 13)
. 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-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 determine this, A549 cells subjected to SAP155 or control siRNA were treated with 20 µM D-erythro-C6 ceramide for 24 h (maximal response time; ref 11
). 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 (11)
, 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. 3
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 (11)
was unaffected by the down-regulation of SAP155 (Fig. 3B
). 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 of the Bcl-x(s) 5' splice site in response to ceramide.
|
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 (12
, 18)
. 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; ref 11
). 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 0.102–0.033 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 0.102–0.012 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 (Table 2
). Furthermore, this sensitization to daunorubicin occurred after only short-term (<48 h) down-regulation of SAP155. In accordance with previous studies (11
, 12
, 22)
, loss of cell viability resulted from the increase in Bcl-x(s) in response to long-term (>60 h) down-regulation of SAP155 (Fig. 4
). Thus, with the use of 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 (11
, 12
, 22)
. 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 (Fig. 4)
.
|
|
DISCUSSION
Previously, our laboratory reported the identification of a CRCE 1 within exon 2 of the Bcl-x pre-mRNA transcript located just upstream of the Bcl-x(s) 5' splicing site (13)
. 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 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. Induction of Bcl-x(s) by RNA interference technology to down-regulate SAP155 lowered the IC50 of the chemotherapeutic agent daunorubicin in a similar manner as ceramide and antisense oligonucleotides targeting the Bcl-x(L) 5' splice site (11
, 12
, 22)
. Finally, ceramide treatment did not induce a loss in cell viability beyond the capacity of SAP155 down-regulation. 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 the signal transduction pathway mediating the 5' alternative splice site selection of Bcl-x pre-mRNA processing, ceramide-induced cell death, and sensitivity to chemotherapeutic agents has been substantiated.
Previously, our laboratory demonstrated that both the dephosphorylation of SR proteins, a family of RNA splicing factors, and activation of the Bcl-x(s) 5' splice site were dependent on the generation of de novo ceramide and the activation of PP1 (11
, 19)
. This led to our initial hypothesis that at least one SR protein isoform of this well-established family of RNA splicing factors was involved in the alternative splicing mechanism of Bcl-x pre-mRNA. Interestingly, these studies did not implicate an SR protein in the regulation of the alternative splice site selection of Bcl-x pre-mRNA as we previously hypothesized. Alternatively, mass spectrometric analysis identified SAP155 as a splicing factor binding to CRCE 1, which was further confirmed by using EMSAs and RNAi technology. Furthermore, down-regulation of SAP155 was shown to regulate the alternative 5' splice site selection of Bcl-x pre-mRNA in the same manner as treatment of cells with ceramide and gemcitabine (GMZ; ref 11
), and ceramide did not effect the alternative 5' splice site selection of Bcl-x when SAP155 was down-regulated. Thus, these data establish SAP155 as a critical splicing factor in the alternative splicing mechanism of Bcl-x via interaction with CRCE 1 and functioning to either repress the Bcl-x(s) 5' splice site or to activate the Bcl-x(L) 5' splice site.
The demonstration that SAP155 regulates the alternative 5' splice site selection of Bcl-x pre-mRNA is very novel as previous studies by Reed et al. (20)
have suggested that SAP155 is an essential splicing factor necessary for specific protein interactions for spliceosomal assembly. Studies in yeast have also identified two homologues to SAP155, Hsh155p and Prp10p, shown to interact with the spliceosome and bind U2AF65 homologues (24, 25). Although these homologues show high conservation to mammalian SAP155 in their C-terminus, the NH2 terminus, which contains key phosphorylation sites and the interaction site for nuclear inhibitor of protein phosphatase-1 (NIPP1), is not conserved, suggesting a more complex role for SAP155 in mammalian cells (26, 27, 28). In the present study, down-regulation of SAP155 only affected the 5' splice site selection of Bcl-x pre-mRNA and had no other effect on the splice variant patterns of other apoptotic pre-mRNAs such as caspase 9 and Bax. Furthermore, down-regulation of human U2AF65, the RNA splicing factor shown to interact with SAP155 in the spliceosome (21)
and also required for yeast viability and growth, had effect neither on the alternative 5' splice site selection of Bcl-x pre-mRNA nor on cell viability within 48 h (unpublished findings). Furthermore, down-regulation of SAP145, 130, and 49, which together with SAP155 comprise the SF3b, an essential complex that associates with U2 snRNP for constitutive splicing activity (17)
, also had no effect on the alternative splicing pattern of Bcl-x. Finally, if SAP155 functioned only in constitutive splicing in mammalian cells, a decrease in both Bcl-x(L) and Bcl-x(s) splice variants would be observed. However, the increase in the Bcl-x(s) splice variant at the expense of Bcl-x(L) as a result of the down-regulation of SAP155 suggests a redirection of the spliceosomal assembly. 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 (29–33). 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, and the main difference between yeast homologues of SAP155 and human SAP155 is the NH2 terminus where nuclear inhibitor of protein phosphatase-1 (NIPP1) associates. Recently, Boudrez et al. (21)
have demonstrated that PP1 is associated with SAP155 via NIPP1. In the same study, they further demonstrated that this interaction is controlled by multisite phosphorylation (21)
; thus, PP1 is in proximity to SAP155 and a regulator of its phospho-state. Recent findings by Tran et al. (22)
also identified SAP155 in association with PP1. 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. 5
).
|
The 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) (12
, 18
, 23)
. Furthermore, the induction of proapoptotic Bcl-x(s) has also been shown to sensitize cells to apoptotic or chemotherapeutic agents (12
, 18)
. In a study by Taylor et al. (12)
, Bcl-x alternative splicing was specifically modulated using an antisense oligonucleotide specific for Bcl-x pre-mRNA at a site surrounding the Bcl-x(L) 5' splice site. Hybridization of this oligonucleotide to Bcl-x pre-mRNA induced an increase in the expression of Bcl-x(s) and a decrease in the expression of Bcl-x(L), resulting in sensitization of the cells to chemotherapeutic agents. Published findings from our laboratory demonstrated that treatment of A549 cells with concentrations of ceramide known to activate the Bcl-x(s) 5' splice site also lowered the IC50 of the chemotherapeutic agent, daunorubicin (11)
. Taken together, these data suggest a link between the signal transduction pathway mediating the 5' splice site selection of Bcl-x pre-mRNA and the sensitivity of cells to apoptosis in response to chemotherapeutics. This study further validated this hypothesis as the use of RNAi to down-regulate the critical regulator of Bcl-x 5' splice site selection, SAP155, also lowered the IC50 of daunorubicin although to a lesser extent than ceramide. Moreover, the down-regulation of SAP155 could not increase the effect of ceramide on lowering the IC50 of DNR. Therefore, these data suggest that the activation of the Bcl-X(s) 5' splice site is a key mechanism within the ceramide signaling pathway as a synergistic or additive effect would demonstrate that SAP155 was in a separate signaling pathway. Since SAP155 siRNA was unable to sensitize A549 cells to DNR to the extent of ceramide, other downstream mechanisms in ceramide signaling may also be involved in this mechanism of sensitization. For example, we demonstrate that the effects of ceramide on caspase 9 alternative splicing remain intact when SAP155 is down-regulated. Thus, the relative contribution of the induction of caspase 9a by ceramide may account for the inability of SAP155 siRNA to sensitize A549 cells to DNR to the same extent as ceramide.
Clearly, the activation of the Bcl-x 5' splice site is important in the loss of viability induced by ceramide. First, only doses of ceramide (>10 µM) that induce the activation of the Bcl-X(s) 5' splice site also induce a significant loss of viability. Second, ceramide could not induce a loss of viability beyond the capacity of the down-regulation of SAP155. These data suggest that the activation of the Bcl-x(s) 5' splice site is a key mechanism in ceramide-induced cell death. Thus, SAP155 may be 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.
In conclusion, these results demonstrate the identification of the RNA trans-factor SAP155 as binding to CRCE 1 of the Bcl-x pre-mRNA transcript and functioning in the regulation of the alternative 5' splice site selection of Bcl-x pre-mRNA. Furthermore, these results also demonstrate that SAP155 is required for the activation of the Bcl-x(s) 5' splice site in response to ceramide. The identification of SAP155 in regulating the alternative splicing mechanism of Bcl-x suggests a novel target of ceramide that will provide a link between ceramide and selection of the Bcl-x(s) 5' splice site. This mechanism of regulation has direct relevance to ceramide-induced cell death and chemotherapeutic sensitivity giving rise to a new target for anticancer therapies.
ACKNOWLEDGMENTS
The mass spectrometric analysis and protein identification were accomplished at the UNC Michael Hooker Proteomics Core Facility, which is partially supported by a gift from an anonymous donor for research targeted to proteomics and cystic fibrosis. The SAP155 Ab was provided by Dr. Robin Reed, Harvard Medical School, Boston, MA. The SRp30a Ab was a gift from Dr. Adrian R. Krainer, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. The hnRNP H and hnRNP F antibodies were supplied by Dr. Doug Black, Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, CA. Dr. Denise Cooper, University of South Florida College of Medicine, Tampa, FL, ifted the SRp30c and SRp40 antibodies. This work was supported by grants from the Veteran’s Administration (Veterans Affairs MERIT Review I to C. E. Chalfant) and from the National Institutes of Health (NIH RO1-HL-072925 to C. E. Chalfant and NIH 1C06-RR-17393 to Virginia Commonwealth University for laboratory renovations).
Received for publication September 16, 2005. Accepted for publication March 31, 2006.
REFERENCES
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