The Bcl-x minigene has been described previously (Massiello, et al., 2004
). The cDNA of human Sam68 was subcloned from pcDNA3-Sam68 (Paronetto, et al., 2003
) into EcoRI–SalI restriction site of pEGFP. Site-directed mutations were inserted by PCR using oligonucleotides containing the mutated residue (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200701005/DC1
). Wild-type and mutated Sam68 cDNAs were also subcloned into pCDNA3-myc eukaryotic expression vector. The human ASF/SF2 cDNA (available from GenBank/EMBL/DDBJ under accession no. NM_006924
) was amplified by RT-PCR using Proofstart polymerase (Pfu; Stratagene) and HEK293 RNA. The cDNA was subcloned into SalI–XbaI restriction sites of p3XFLAG (Sigma-Aldrich). GFP-Sam68-RS was generated by fusing the RS domain of ASF/SF2 (aa 194–248) to the C terminus of Sam68. The cDNA encoding the RS domain of ASF/SF2 was amplified by PCR and subcloned into SalI–BamHI restriction sites of pEGFP in fusion with Sam68 upstream of the TAA codon. All cDNAs used in the experiments were sequenced by Cycle Sequencing (BMR Genomics). Expression vector for Sam68m1, containing the phosphorylation mutated sites, was provided by H. Konig (Institut für Toxikologie und Gnetik, Karlsruhe, Germany).
Cell cultures and transfections
HEK293 cells were maintained in DME (Invitrogen) supplemented with 10% FBS (BioWhittaker Cambrex Bioscience), penicillin, and streptomycin. For transfections, HEK293 cells were plated in 35-mm dishes 1 d before and transfected with 1 μg of DNA (Bcl-x minigene, pEGFP-Sam68wt, pEGFP-Sam68V229F, pEGFP-Sam68NLSKO, pEGFP-Sam68RS, pFLAG-Asf/SF2, pCMV5-Fyn, pCMV5-FynY528F, pCDNA3-MEKK1, pCDNA3-myc-Sam68, pCDNA3-myc-Sam68V229F) using lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. At 24 h after transfections, cells were collected for RNA or biochemical analysis. For RNAi, cells at ~50/60% confluence were transfected with siRNAs (MWG Biotech) using Oligofectamine and Opti-MEM medium (Invitrogen). Transfections were performed for 2 or 3 consecutive days. Sequences for Sam68 and hnRNP A1 siRNAs are listed in Fig. S4. Transfection of antisense Bcl-x(L) oligonucleotide (Calbiochem) was performed as for RNAi using Oligofectamine.
Extraction of RNA and proteins from cultured cells
Total RNA was extracted from transfected HEK293 cells using cold Trizol reagent (Invitrogen), according to the manufacturer's instructions. RNA was resuspended in RNase-free water (Sigma-Aldrich) and immediately frozen at −80°C for further analysis.
For protein extraction, HEK293 cells were resuspended in lysis buffer (100 mM NaCl, 10 mM MgCl2, 30 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 10 mM β-glycerophosphate, 0.5 mM NaVO4, and protease inhibitor cocktail [Sigma-Aldrich]) supplemented with 0.5% Triton X-100. The extracts were centrifuged for 10 min at 12,000 g at 4°C, and the supernatants were collected and used for Western blot or immunoprecipitation experiments.
HEK293 cells were homogenized in lysis buffer (100 mM NaCl, 10 mM MgCl2, 30 mM Tris-HCl, 1 mM DTT, protease inhibitor cocktail, and 40 U/ml RNase OUT [Invitrogen]) supplemented with 0.5% Triton X-100. Soluble extracts were separated by centrifugation at 10,000 g for 10 min, and they were precleared for 2 h on protein A–Sepharose beads (Sigma-Aldrich) in the presence of 2 μg rabbit IgGs, 0.05%BSA, and 0.1 μg/ml yeast tRNA. After centrifugation for 1 min at 1,000 g, supernatants were incubated with 2 μg anti-Sam68 (Santa Cruz Biotechnology, Inc.), anti-GFP (Roche), or rabbit IgGs for 3 h at 4°C under constant rotation. Beads were washed three times with lysis buffer, and an aliquot was eluted in SDS sample buffer for Western blot analysis. The remaining beads were incubated with lysis buffer in the presence of (RNase-free) DNase (Roche) for 15 min at 37°C and washed three times with lysis buffer before incubation with 50 μg proteinase K (Roche) for an additional 15 min at 37°C. Coprecipitated RNA was then extracted by standard procedure and used for RT-PCR using BclX-1 and rtBclX-2 primers (Fig. S4). For the coimmunoprecipitation experiment with hnRNP A1, nuclear extracts were prepared by resuspending cells in ipotonic buffer (10 mM Tris/HCl, pH 7.4, 10 mM NaCl, 2.5 mM MgCl2, 1 mM DTT, protease inhibitor cocktail, 30 U/ml RNase OUT, 10 mM β-glycerophosphate, and 0.5 mM NaVO4). After incubation on ice for 7 min, samples were centrifuged at 700 g for 7 min. Pelleted nuclei were resuspended in ipotonic buffer supplemented with 90 mM NaCl and 0.5% Triton X-100, sonicated, and stratified on 30% sucrose. After a centrifugation at 5,000 g for 15 min, nuclear extracts were precleared and immunoprecipitated as described. Where indicated, nuclear extracts were treated with 100 μg/ml RNase A (Sigma-Aldrich).
GST pull-down assay of the mRNA–Sam68 and protein–protein interactions
For protein–protein interactions, GST and GST-Sam68 were purified from Escherichia coli
as previously described (Sette et al., 1998
) and incubated for 1 h with nuclear extracts. For RNA–protein interactions, 2 μg of purified GST proteins were equilibrated for 1 h in 50 mM Tris-HCl, pH 7.4, 100 mM KCl, 2 mM MgCl2
, 1 mM EDTA, 1 mM DTT, 40 U/ml RNase OUT, and 0.2% Nonidet-P40 supplemented with 0.05% BSA and 0.1 μg/ml yeast tRNA. Purified total RNA from HEK293 was added to the beads and incubated at 4°C under constant rotation. Beads were washed and RNA was extracted as described in the previous paragraph.
1 μg of RNA from HEK293 transfected cells or all of the coimmunoprecipitated RNA was used for RT-PCR using M-MLV reverse transcriptase (Invitrogen) according to manufacturer's instructions. 10% of the reverse-transcription reaction was used as template together with the following primers: endogenous Bcl-x, Bcl-X-1 (forward) and Bcl-X-2 (reverse); real-time PCR, rt-BclX-1 (forward) and rt-BclX-2 (reverse); Bcl-x minigene, mg-BclX-1 (forward) and mg-BclX-2 (reverse). All primer sequences are listed in Fig. S4. Real-time PCR was performed using the iQ Sybr-green Supermix (Bio-Rad Laboratories) according to manufacturer's instructions.
Western blot analysis
Cell extracts or immunoprecipitated proteins were diluted in SDS sample buffer and boiled for 5 min. Proteins were separated on 10% SDS-PAGE gels and transferred to Hybond-P membranes (GE Healthcare) as previously described (Sette et al., 2002
). The following primary antibodies (1:1,000 dilution) were used (overnight at 4°C): rabbit anti-Sam68, rabbit anti-Erk2, rabbit anti-Fyn, and mouse anti-phosphotyrosine (PY20; Santa Cruz Biotechnology, Inc.); mouse anti-ASF/SF2 (US Biological); mouse anti–hnRNP A1, mouse anti-tubulin, and rabbit anti-actin (Sigma-Aldrich); mouse anti–hnRNP H/F (Abcam); rabbit anti–phospho-ERKs (Cell Signaling); rabbit anti-GFP (Roche); rabbit anti-phosphoserine and anti-phosphothreonine (Stressgen); and rabbit anti–Bcl-x (BD Biosciences). Secondary anti-mouse or anti-rabbit IgGs conjugated to horseradish peroxidase (GE Healthcare) were incubated with the membranes for 1 h at room temperature at a 1:10,000 dilution in PBS containing 0.1% Tween 20. Immunostained bands were detected by chemiluminescent method (Santa Cruz Biotechnology, Inc.).
Annexin V staining
Transfected cells grown on 35-mm plates were harvested and processed for annexin V staining or for Western blot analysis. For annexin V staining, cells were washed in PBS and stained with the annexin V–PE (BD Biosciences) according to the manufacturer's instructions. GFP-positive cells were then analyzed with a FACSCalibur Flow Cytometer (Becton Dickinson).
Transfected HEK293 cells were fixed in 4% paraformaldehyde and washed three times with PBS. Cells were permeabilized with 0.1% Triton X-100 for 7 min and incubated for 1 h in 0.5% BSA. Cells were washed three times with PBS and incubated for 2 h at room temperature with antibodies against cleaved casp3 (1:400; Sigma-Aldrich), SC35 (1:200; Sigma-Aldrich), Asf /SF2 (1:100), or hnRNP A1 (1:400), followed by 1 h of incubation with cy3-conjugated anti-mouse IgGs (Chemicon). After washes, slides were mounted with MOWIOL reagent (Calbiochem) and analyzed by confocal microscopy using an inverted microscope (Carl Zeiss MicroImaging, Inc.).
Image acquisition and manipulation
Images in , , , and Fig. S1 A were taken from an inverted microscope (IX70; Olympus) using an LCA ch 20×/0.40 objective. Images in were taken from a microscope (Axioskop; Carl Zeiss MicroImaging, Inc.) using a Pan-Neofluar 40×/0.75 objective. Images were acquired at room temperature using a RT-slider camera (Diagnostic Instruments) and the IAS2000 software (Biosistem82; Delta Sistemi). The confocal images in and Fig. S3 were taken from a confocal microscope (LSM510; Carl Zeiss MicroImaging, Inc.) using a Plan-Neofluar 40×/1.3 oil differential interference contrast objective and the LSM510 software (Carl Zeiss MicroImaging, Inc.). Images were acquired as TIFF files, and Photoshop and Illustrator (Adobe) were used for composing the panels.
Online supplemental material
Fig. S1 shows that the ERK1/2 pathway does not regulate Sam68-mediated alternative splicing of Bcl-x. Fig. S2 shows the effects of overexpressed Fyn on the alternative splicing of endogenous Bcl-x transcripts. Fig. S3 shows the localization of Sam68 mutants and splicing regulators in HEK293 cells. Fig. S4 shows a list of the oligonucleotides used for the PCR reactions in the article. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200701005/DC1