Table 5. Annotated as described in Table 1. SF2/ASF target exons located adjacent to alternative cassette exons Gene ID NETO2 PRKRA RPS24 PABPC1 BRD2 YWHAH HNRPDL HNRPAB MPZL1 Exon Position 12 13 9 4 6 2 9 8 6 Gene Description neuropilin (NRP) and tolloid (TLL)-like 2 protein kinase, interferon-inducible double stranded Cytoplasm RNA dependent activator ribosomal protein S24 poly(A) binding protein, cytoplasmic 1 bromodomain containing 2 tyrosine 3-monooxygenase/tryptophan 5- monooxygenase activation protein, eta polypeptide heterogeneous nuclear ribonucleoprotein D-like heterogeneous nuclear ribonucleoprotein A/B myelin protein zero-like 1 Cytoplasm Nucleus; Polysome Nucleus Nucleus; Cytoplasm Nucleus Nucleus Kinase Other kinase Other Other Other Localization Cytoplasm Gene Family Function Transmembrane Protein development; neuronal function Ribosome Component Translation factor SF2/ASF RNA Targets Pro-apoptosis translation regulator generation of spermatozoa transcription regulator mRNA processing mRNA processing Nucleus; Cytoplasm Transmembrane Protein Receptor doi:10.1371/journal.pone.0003369.t005 SF2/ASF for in vitro evolution of binding sites [19]. By contrast, functional SELEX experiments, in which bona fide ESEs within reporter constructs are replaced by randomized sequence and in vitro splicing is driven by a single recombinant SR protein in S100 complementation assays, yielded different consensus motifs for SF2/ASF [18]. The functional SELEX motifs are more heterogeneous in nature but may represent binding sites for SF2/ASF that can lead to productive splicing. The differences between motifs identified by functional SELEX and CLIP may reflect inherent differences in the experimental conditions. For example, photo cross-linking of protein-RNA interactions is believed to be fairly inefficient, but it allows for recovery of sequences under stringent conditions. It remains possible that sequences identified by CLIP represent only a subset of possible binding sites that are both abundant and suitable for cross-linking. Data presented in Fig. 4B demonstrates that SF2/ASF can regulate the alternative splicing of a subset of pre-mRNAs suggesting that the observed binding sites are functionally relevant. Indeed, 26 out of 44 endogenous pre-mRNAs containing binding sites for SF2/ASF within or proximal to cassette exons appear to be functionally relevant when tested by RT-PCR (Fig. 4B and data not shown). Thus we feel the CLIP method has captured a subset of biologically relevant SF2/ASF binding sites. Cataloguing the collection of cis-acting elements directing post- transcriptional gene regulation on a global scale is only at the initial stages of investigation. In silico studies have successfully elucidated cis-acting elements that are enriched or co-evolve with constitutive and alternative exons. Several recent computational screens have defined thousands of hexa- or octamers as splicing regulatory sequences [32–34]. If a combinatorial code governing splice site selection exists, the solution will likely emerge through interdisciplinary approaches merging computational methods with biochemical and functional genomics approaches. Methods such as SELEX, CLIP and RIP-Chip have the potential to illuminate the specificity and functions of RNA binding proteins by making connections between trans-acting RNA binding proteins and the biological processes they regulate [13,35–37]. CLIP also provides the added value of determining potential mechanisms of RNA binding protein action by revealing the positional context of cis- acting RNA elements within a transcript [13]. RNA binding protein target specificity can also be evaluated using platforms to profile alternative splicing such as exon tiling arrays, spliced junction array platforms and RNA-Seq [38–40]. Discrimination of direct versus indirect effects is a limitation of these experiments however this may be overcome by applying computational models of consensus binding sites generated by CLIP or SELEX to classify global changes in alternative splicing [39,41]. Our initial CLIP experiment with SF2/ASF revealed just a small snap shot of putative RNA targets. One clear limitation of this study is the inefficiency of the Sanger sequencing. Merging CLIP with high throughput genome wide sequencing platforms such as Solexa and 454 (Illumina, USA and Roche Diagnostics, USA, respectively), will vastly expand our ability to define the RNA binding specificity. These data will allow for a high resolution map of SF2/ASF binding sites that not only illuminate mechanisms of action in pre-mRNA splicing but also reveal connections to important biological processes such as genome stability, cellular transformation and non-coding RNA processing. Materials and Methods Cell culture and transient transfection For CLIP analysis of SF2/ASF, HEK293T cells were grown to 75–80% confluence at 37uC, 5% CO2. HEK293T cells were cultured in DMEM containing 10% Fetal Calf Serum and antibiotics. Transient transfection of HEK293T cells with plasmid DNA was performed using Lipofectamine 2000 (Invitrogen) following the manufacturers instructions. Transient transfection of siRNA duplexes and plasmid DNA were performed using Duo-fect transfection reagent (Dharmacon) following the manufacturers specifications. Cells were harvested 48 hours following transfection. siRNA transfection efficiency was monitored using siGLO green siRNA duplexes (Dharmacon) and was found to be 80–95% efficient. Plasmids and siRNA T7 Epitope tagged SF2/ASF was expressed from the pCGT7 vector and has been described previously [42]. siRNA Smartpools targeting the endogenous SF2/ASF transcript, or scrambled nontargeting siRNA duplexes were purchased from Dharmacon. Cross-linking Immunoprecipitation of SF2/ASF from HEK293T cells CLIP analysis of SF2/ASF was performed as described by Ule et al.[32] with the following modifications relating to extract PLoS ONE | www.plosone.org 7 October 2008 | Volume 3 | Issue 10 | e3369