SF2/ASF RNA Targets Figure 2. Distribution of SF2/ASF binding sites in relation to gene structure and sub-cellular localization. A) The ‘‘coding’’ SF2/ASF CLIP tags from nuclear, cytoplasmic and polysomal sets were analyzed in relation to their relative position within genes. This showed that the majority map to intronic or to coding exonic sequences and only a small proportion mapped to UTRs. B) Venn diagram of exonic SF2/ASF binding sites from Nuclear, cytoplasmic and polysomal sub-cellular fractions. The number of unique binding sites identified in each fraction is designated as ‘‘n’’. doi:10.1371/journal.pone.0003369.g002 Identification of a consensus binding site for SF2/ASF The in vitro RNA binding specificity of many SR proteins including SF2/ASF, have been well-characterized using approaches such as selected evolution of ligands by exponential enrichment (SELEX) [17]. Different consensus sites for SF2/ASF have been obtained using different SELEX strategies [18,19]. We used the motif finding algorithm MEME, to search exonic or intronic RNA fragments for over represented sequences [20,21]. Fig. 3A shows that a purine-rich consensus motif is present within exonic sequences targeted by SF2/ASF with a highly significant expectation value (e value) of 3.861024. No other statistically significant motifs were identified within the exonic sequences. By contrast MEME was unable to detect an enriched sequence motif within the intronic pool of binding sites identified by CLIP. These data suggest that intronic binding sites may prove to be a nonspecific contaminant. To test the validity of the consensus model, we asked if the consensus sequence is enriched in exonic or intronic sequences across the genome using composite exons [18]. We then used the position weight matrix calculated from the consensus motif to determine the number of binding sites at each position across all intron/exon/intron composite sequences. Fig. 3B demonstrates that the consensus motif for SF2/ASF is highly enriched at the edges of exons relative to intronic sequences or the interior of exon sequences. These data support the hypothesis that SF2/ASF plays a role in the establishment of exon identity. Annotation of alternatively spliced pre-mRNA targets of SF2/ASF SR proteins have well characterized roles in both constitutive and alternative pre-mRNA splicing. Resolving these dual functions of SR proteins solely by the use of heterologous substrates and mini-gene reporters has proven difficult. In order to determine if the endogenous binding sites for SF2/ASF may reveal any clues to the roles of SR proteins in alternative splicing we manually curated annotated ENSEMBL transcripts targeted by SF2/ASF for examples of alternative splicing using two distinct databases: AceView and FAST-DB [22,23]. Of the 234 exonic binding sites identified by CLIP, only 72 were subject to some form of alternative processing (Table 1–4 for details on binding sites associated with specific types of alternative splicing). We also identified 9 binding sites within constitutive exons located downstream and adjacent to alternative cassette exons (See Table 5). This type of arrangement suggests that SF2/ASF may promote skipping of the upstream cassette exon, as recently demonstrated for alternative splicing of the receptor tyrosine kinase RON during breast cancer metastasis [24]. Within this list of alternative exons targeted by SF2/ASF there is a modest enrichment for genes encoding RNA binding proteins (SFRS1, PABPC1, hnRNPDL, hnRNPAB, SERBP1, RPL5, SON, RPL0, Figure 3. Identification of the SF2/ASF consensus binding site and its frequency distribution in coding exons and flanking introns. A) The exonic or intronic clip tags were analyzed using MEME to identify sequence patterns that are over-represented. MEME was unable to identify a consensus motif within the intronic binding sites. B) This pattern was used to analyze composite sequences made for all exons with more than 100 nucleotides, from protein coding genes. This analysis shows that the pattern is over-represented within exonic sequences and in particular within the exon 59 prime and middle of the exons. doi:10.1371/journal.pone.0003369.g003 PLoS ONE | www.plosone.org 3 October 2008 | Volume 3 | Issue 10 | e3369