Figure 4. Alternative splicing of endogenous RNA transcripts targeted by SF2/ASF. A) Western blot analysis of HEK293T cells co- transfected with siRNA duplexes and expression plasmids. The upper panel is probed with anti-SF2/ASF monoclonal antibody, the lower panel is probed with anti-GAPDH monoclonal antibody. Lane 1, non transfected control (NTC); Lane 2, Mock (no siRNA or plasmid); Lane 3, siRNA targeting SF2/ASF and empty vector (pCGT7); Lane 4, scrambled siRNA and empty vector; Lane 5, scrambled siRNA and T7-tagged SF2/ASF expression plasmid (pCGT7 SF2). Total RNA was purified for RT-PCR analysis from samples corresponding to lanes 3 and 5. B) RT-PCR analysis of alternative splicing. Radiolabeled products were visualized by phosphorimaging. Schematic diagrams show exon-intron structure of target genes and patterns of alternative splicing. Thin rectangles indicate untranslated regions, thick rectangles indicate coding regions, thick lines indicate introns and thin lines indicate the alternative splicing event. Exons identified by CLIP are yellow. The grey region in the SFRS1 39UTR indicates a retained intron. Arrowheads to the left of each panel indicate the included (gray arrowhead) and skipped (black arrowhead) alternative mRNA isoform. Lane 1 and 2 contain RNA purified from SF2/ASF overexpressing and depleted cells, for example see panel A, lanes 5 and 3, respectively. The sub-cellular fraction corresponding to the initial CLIP experiment is indicated below each gene symbol. doi:10.1371/journal.pone.0003369.g004 preparation and RNase treatment. Nuclear and cytoplasmic extracts were prepared from UV-treated or control cells as previously described [7]. The soluble extract was treated with SF2/ASF RNA Targets 30 U RQ DNase 1 for 20 min at 37uC. The reactions were terminated by the addition of 20 mM EDTA. Subsequently, ribosomal subunits were cleared by centrifugation of the extract at 100,0006 g using an Optima Max ultracentrifuge (Beckman Coulter, USA) in a TLA120.2 rotor for 20 min. Cleared extracts were then treated with a dilute cocktail of RNase A/T1 (Ambion, USA) at a final dilution range of 1:1000–1:10,000 for 20 min at 37uC. 200 U RNaseOut (Invitrogen, USA) was then added to the extract. Proteins were then partially denatured by addition of an equal volume of buffer A (26 PBS, 0.2% SDS, 1% NP-40). An aliquot of each UV-treated extract was used to prepare input RNA fragments. The remainder of the extract was used for immuno- precipitation with anti-SFRS1 monoclonal antibody. Characterization of the CLIP tags All Human exon and gene information was downloaded from Ensembl, using the BioMart tool. The sequence data was downloaded using Perl scripts to access the ensembl database through the Ensembl Core Perl API. The CLIPS sequences were mapped against unspliced gene, cDNA and coding sequences using Mega BLAST (Zhang et al. 2000). The unambiguous mapping of the CLIP tags allowed us to match the CLIPs to Ensembl gene ids. The characterization of type of splicing event that occurs on the alternatively spliced exons that were found to be bound by SF2/ASF was performed according to Ensembl exon information. Identification of the SF2/ASF binding site The distinct CLIP tags that match exons were used on the MEME (Bailey et al., 2006) web server (http://meme.sdsc.edu/ meme/meme.html) to identify patterns that are probabilistically significant. The intronic set of CLIP tags was used in the same way but did not result in any meaningful pattern. The genome wide distribution of the SF2/ASF binding site pattern To identify the genome-wide distribution of SF2/ASF binding sites around exon splice sites, for each exon from a protein coding gene (greater than 100 bp), we generated a composite sequence made of the 75 bp upstream of the 39 splice site, the 59 exonic 25 bp, the 50 bp from the middle of the exon, the 25 bp 39 exonic and 75 bp downstream of the 59 splice site. There were in total 174,440 exons that satisfied this criteria. These were then analyzed using the binding sites in CLIP tags that were identified by MEME. Parsing of Data All parsing of data was performed using multiple Perl scripts. in vivo splicing assay HEK293T cells were transfected as described above. Cells were harvested 48 hours post-transfection. One aliquot was used for western blot analysis the other was used to prepare total RNA from the cytosolic fraction. RNA was extracted using TRI Reagent LS (Sigma) following the manufacturers instructions. cDNA was synthesized using oligo dT primer and Superscript RT III (Invitrogen). 50 ng of cDNA was used as a template for RT- PCR analysis. Primers were 59 end labelled with 32PcATP and T4 PNK (NEW England Biolabs). Primers are available upon request. PCR was performed using the following conditions: 3 min at 94uC; 30 cycles of 94uC 30 sec, 59uC 30 sec, 72uC 60 sec; 72uC 5 min. Following PCR, the reactions were ethanol precipitated and resolved upon 10% polyacrylamide/7M urea gels. RT-PCR reactions were visualized by autoradiography or phosphorimager. PLoS ONE | www.plosone.org 8 October 2008 | Volume 3 | Issue 10 | e3369