Gene Family enzyme other other enzyme kinase kinase other other transcription regulator Nucleus; Cytoplasm; Polysome transporter Nucleus; Cytoplasm Nucleus; Cytoplasm; Polysome other Nucleus transcription regulator transcription regulator Table 1. Annotation of alternative cassette exons bound by SF2/ASF. Partial list of alternative cassette exons bound by SF2/ASF Gene ID ACIN1 BECN1 CALM2 DHX15 ERCC6 MAPK3 NUP43 PIP5K2A SON TCF12 WDR42A Y13871 ZHX1 12 11 4 5 2 6 4 7 3 9 1 3 Exon Position Gene Description apoptotic chromatin condensation inducer 1 beclin 1 (coiled-coil, myosin-like BCL2 interacting protein) calmodulin 2 (phosphorylase kinase, delta) DEAH (Asp-Glu-Ala-His) box polypeptide 15 excision repair cross-complementing rodent repair deficiency, complementation group 6 mitogen-activated protein kinase 3 Cytoplasm nucleoporin 43kDa phosphatidylinositol-5-phosphate 4-kinase, Nucleus type II, alpha SON DNA binding protein transcription factor 12 (HTF4, helix-loop- helix transcription factors 4) Nucleus Cytoplasm Localization Nucleus Nucleus; Polysome Cytoplasm Cytoplasm Cytoplasm 11 WD repeat domain 42A hypothetical protein LOC202181 zinc fingers and homeoboxes 1 SF2/ASF RNA Targets Function apoptotic chromosome condensation anti-apoptosis;cellular defense response Calcium Binding;G-protein coupled receptor protein signaling pathway mRNA processing transcription-coupled nucleotide- excision repair intracellular signaling cascade nuclear envelope glycerophospholipid metabolic process anti-apoptosis regulation of transcription from RNA polymerase II promoter Unknown Unknown negative regulation of transcription, DNA-dependent Exons were annotated manually according to AceView and FAST-DB databases. Column Headers: Gene ID, official gene symbol; Exon position, number of exon within the primary transcript; Gene Description; Localization, the sub-cellular fraction used for CLIP experiment; Gene Family; Function, the primary encoded function of gene. This table is a partial list of cassette exons bound by SF2/ASF. The complete list can be found in Table S1. doi:10.1371/journal.pone.0003369.t001 RPS10, RPL12, RPS24, RPL19, PKRA) and genes involved in regulation of biological processes such as cell division, proliferation and apoptosis (CDK4, MAPK3, ACIN1). The ENSA gene is also of interest as it is bound by SF2/ASF in each sub-cellular compartment and is a Type 2 diabetes candidate gene. ENSA is an endogenous ligand for the sulfonylurea receptor which plays an important role in insulin release from pancreatic beta cells [25,26]. Several of the alternative splicing events involving exons targeted by SF2/ASF lead to premature termination codons and potentially NMD (SFRS1, CDK4, MAPK3, for example) however the majority of alternative events alter the primary structure of the encoded polypeptide (not shown). All of these splicing events can be visualized using the Friendly Alternative Splicing and Transcript Diversity database (http://www.fast-db.com; [22]). Regulation of endogenous nuclear and cytoplasmic mRNA processing by SF2/ASF A majority of RNA fragments that co-purified with SF2/ASF contain a purine-rich consensus motif, reminiscent of previous SELEX binding sites [19]. Interestingly, a subset of mRNA targets was bound by SF2/ASF in both the nucleus and the cytoplasm. Three of these targets, ENSA (endosulfine a), PABPC1 (poly A binding protein 1) and NETO2 (neuropillin-like tolloid receptor 2) contained binding sites for SF2/ASF within or flanking alternative cassette exons, whereas the SFRS1 (pre-mRNA encoding SF2/ ASF) contains a binding site adjacent to a retained intron. We then asked whether SF2/ASF modulates the alternative splicing of these pre-mRNAs in the nucleus and enhance their translation in the cytoplasm. We manipulated the levels of SF2/ASF in HEK293T cells by transfecting cells with siRNA targeting endogenous SF2/ASF, scrambled control siRNA or plasmid to increase levels of SF2/ASF by transient ectopic expression and assayed alternative splicing of endogenous ENSA, SF2/ASF, PABP and NETO2. Fig. 4A shows a Western blot from control cells (lanes 1,2, and 4, respectively) or cells depleted or over expressing SF2/ ASF (lanes 3 and 5, respectively). In the context of the ENSA pre- mRNA, the observed binding site is located within an alternative cassette exon. As expected ectopic expression of SF2/ASF increases inclusion of the alternative exon, relative to the cells depleted for SF2/ASF (Fig. 4B). Binding sites for SF2/ASF within the PABP and Neto2 pre-mRNAs are in exons proximal to alternative cassette exons. We found that ectopic expression of SF2/ASF causes increased skipping of the alternative cassette exon in both cases (Fig. 4B). Finally, the observed binding site for SF2/ASF within its’ own pre-mRNA (transcribed from the SFRS1 gene) is located near an ultra-conserved element adjacent to a retained intron within the 39UTR [27,28]. This intron is believed to attenuate expression of the SFRS1 gene through regulated unproductive splicing and translation (RUST) mechanism [27]. We observe that ectopic expression of SF2/ASF increase splicing of this intron and provides supporting evidence for the RUST hypothesis (Fig. 4B). The splicing of each of these endogenous pre-mRNA targets is effected by increasing the intracellular concentrations of SF2/ASF. It is known that SF2/ASF can remain associated with mRNA following the splicing reaction and that SF2/ASF can enhance the translation of reporter mRNAs [7,8]. The CLIP method revealed that a subset of SF2/ASF mRNA targets, including PABP, NETO2, ENSA and SFRS1, were bound by SF2/ASF in the PLoS ONE | www.plosone.org 4 October 2008 | Volume 3 | Issue 10 | e3369