revealing genomic locus of the RNA as well as the position of the RNA-protein interaction within the RNA molecule. CLIP analysis of the neural specific splicing factor Nova identified a post- transcriptional regulatory network related to synaptic function. Mechanistic studies determined the relationship between positions of Nova binding sites and effects on splice site selection [13,14]. These data were used to build an RNA map capable of predicting Nova-dependent splice site selection [15]. Here, we use CLIP to identify RNA targets for the shuttling SR protein, SF2/ASF, in order to test the hypothesis that this SR family protein member regulates nuclear and cytoplasmic expression of specific mRNAs in living cells. This sampling of nuclear, cytoplasmic and polyribo- some-associated mRNA targets suggests that SF2/ASF may remain associated with a subset of specific transcripts as they are trafficked from the nucleus to the cytoplasm. Furthermore we provide evidence that SF2/ASF couples alternative splicing with enhanced translation for several endogenous mRNAs. These data provide insights to the targets and roles of an essential shuttling RNA binding protein in coupling nuclear and cytoplasmic steps of post-transcriptional gene expression. Results Cross-linking immunoprecipitation of SF2/ASF Previously, we have shown that SF2/ASF binds directly to cytoplasmic mRNA in vivo and enhances the translation of reporter mRNAs both in vitro and in vivo [8], suggesting that SF2/ASF may regulate the nuclear and cytoplasmic fate of specific endogenous mRNAs. Despite nearly two decades of study, few endogenous targets of SR proteins have been identified. In order to determine if SF2/ASF can regulate nuclear and cytoplasmic processing of endogenous mRNAs we used the CLIP protocol to identify binding sites for SF2/ASF in mRNAs from different subcellular fractions. Nuclear, cytoplasmic and polyribosome-containing sub- cellular fractions were prepared as previously described [7] from control cells or those exposed to UV irradiation and cell extracts were treated with decreasing amounts of RNase A/T1 to partially degrade cross-linked RNA molecules. SF2/ASF was immunopre- cipitated from each extract under stringent conditions. Purified SF2/ASF-RNA complexes were modified on beads by the SF2/ASF RNA Targets addition of a 39 RNA linker and 32P at the 59 end. Complexes were resolved on 10% Novex Nupage gels (Invitrogen, USA) and transferred to nitrocellulose. Fig. 1 shows UV-dependent, RNase- sensitive complexes immunoprecipitated by the anti-SF2/ASF monoclonal antibody. Note the increased molecular weight of the complex as RNase concentrations are decreased. The heteroge- neous migration of these complexes is due to increasing length of captured RNA fragments. Complexes between 37 and 42 kDa were purified from nitrocellulose membranes. RNA was extracted, ligated to a 59 RNA linker and amplified by RT-PCR (not shown). Amplicons were then concatamerized, ligated in pcDNA3.1, cloned and sequenced. Mapping of SF2/ASF mRNA targets We obtained sequences from more than 3,500 amplicons corresponding to putative SF2/ASF RNA targets. We used BLAT to align sequences to the human genome allowing for two mis- matches or gaps within the alignment [16]. For this analysis we focused on RNA fragments corresponding to protein coding genes, any other sites were ignored. 1,237 amplicons could be mapped to a total of 326 positions in 180 protein coding genes; 43% of the binding sites were represented by multiple sequences. A majority of binding sites (68%) fell within exonic sequences (Fig. 2A). We also detected binding sites for SF2/ASF within intronic sequences. However, as shown below, we were unable to detect a consensus motif within the pool of intronic binding sites, therefore we focused the remaining analysis on the exonic pool of sequences. We compared the binding specificity of SF2/ASF RNA targets identified in different sub-cellular compartments in order to determine if any RNA transcripts were in common. We found that 72 exonic binding sites are engaged by SF2/ASF in both the nucleus and the cytoplasm or polysome fraction. Interestingly, 15 binding sites in 12 different genes are engaged by SF2/ASF in all three compartments (Fig. 2B). These data provide strong evidence that SF2/ASF associates with a subset of messenger RNAs in the nucleus and that the same cis-acting element is recognized in the cytoplasm. We hypothesize that SF2/ASF remains bound to the mRNA as the mRNA is trafficked from the nucleus to the cytoplasm [7]. Figure 1. Cross-linking immunoprecipitation of SF2/ASF from different HEK293T cellular fractions. SF2/ASF-RNA complexes, indicated by the bracket, were visualized by autoradiography. Positions of molecular weight standards are given at the left of each panel and exposure of cells to ultra violet radiation (UV) is indicated by + or 2. doi:10.1371/journal.pone.0003369.g001 PLoS ONE | www.plosone.org 2 October 2008 | Volume 3 | Issue 10 | e3369