Localization Gene Family Function transmembrane receptor insulin receptor signaling pathway hematological and neurological expressed 1 insulin-like growth factor 1 receptor PRELI domain containing 1 calmodulin 2 (phosphorylase kinase, delta) Cytoplasm S-adenosylhomocysteine hydrolase zinc finger protein 91 homolog (mouse) ubiquitin protein ligase E3B ribosomal protein L10a glycerol kinase ribosomal protein S10 ribosomal protein L19 N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucos-aminidase beta-2-microglobulin ethanolamine kinase 1 Not Determined nuclear receptor subfamily 2, group C, member 1 PWP1 homolog (S. cerevisiae) centromere protein O transcription factor 12 Nucleus Nucleus Nucleus Nucleus Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm Nucleus Nucleus Nucleus Nucleus Cytoplasm microsomal glutathione S-transferase 3 Nucleus; Cytoplasm enzyme eukaryotic translation elongation factor 1 alpha 1 Nucleus; Cytoplasm; translation Polysome Nucleus; Cytoplasm Nucleus; Cytoplasm Nucleus; Cytoplasm other enzyme Nucleus; Cytoplasm transmembrane receptor immune response Cytoplasm; Polysome kinase other other other other enzyme enzyme other kinase other transcription regulator Unknown transcription regulator other other transcription regulator SF2/ASF RNA Targets glycosphingolipid metabolic process; lipid transport Not Determined multicellular organismal development Calcium Binding; G-protein coupled receptor protein signaling pathway one-carbon compound metabolic process Protein degredation Ribosome Component Ribosome Component lipid metabolic process; signal transduction Ribosome Component carbohydrate metabolic process; lysosome organization and biogenesis Phosphatidylethanol-amine biosynthetic process translational elongation Unknown Unknown Unknown role in cell growth and/or transcription Regulation of transcription by RNA Polymerase II Table 2. Annotated as described in Table 1. SF2/ASF target exons containing alternative 39 splice sites Exon Position Gene Description prosaposin Gene ID PSAP HN1 IGF1R PRELID1 CALM2 AHCY ZFP91 UBE3B RPL10A GK RPS10 MGST3 RPL19 NAGPA B2M ETNK1 EEF1A1 FLJ00313 NR2C1 PWP1 CENPO TCF12 7 3 5 3 21 10 11 28 6 17 3 2 6 5 2 2 3,6 7 12 13 5 9 doi:10.1371/journal.pone.0003369.t002 cytoplasm of HEK293T cells. This observation suggests that SF2/ ASF may also enhance the translation of these particular mRNA targets. To test this hypothesis we asked whether a specific mRNA target of SF2/ASF exhibited increased association with polyribo- somes, the actively translating pool of ribosomes, when SF2/ASF is overexpressed. Cytoplasmic extracts were prepared from HEK293T cells transfected with empty expression vector or epitope tagged SF2/ASF (Fig. 5A). Cytoplasmic extracts were resolved across 10–45% sucrose gradients. Following fraction- ation, total RNA was extracted from every other fraction and cDNA was synthesized using oligo dT primer. Upon ectopic expression of SF2/ASF all four endogenous mRNA targets showed increased association with polyribosomes (Fig. 5A, lanes 5–8). By contrast, levels of GAPDH remain constant across the gradient when SF2/ASF is overexpressed. We also examined the distribution of several other endogenous targets, but found no difference when SF2/ASF is over expressed, suggesting that this effect is specific for a subset of transcripts rather than a global phenomenon (data not shown). Additionally, there are no obvious differences between the distributions of total RNA across the gradient. These data are in good agreement with our previous findings that SF2/ASF can enhance the polysome association and translation of luciferase reporter mRNAs both in vitro and in vivo [7–9]. Finally these data provide clear evidence that SF2/ASF can drive enhaced polysome association of endogenous mRNAs in vivo. Discussion SF2/ASF is a nucleo-cytoplasmic shuttling RNA binding protein with clearly defined activities in spliceosome assembly. Our previous work established a role for SF2/ASF in mRNA translation [7,8]. These findings suggested that SF2/ASF may co- ordinate the nuclear and cytoplasmic steps of post-transcriptional gene expression for a subset of pre-mRNAs. This hypothesis is further supported by the observation that extrinsic signals leading to activation of the AKT protein kinase enhance the alternative splicing and translation of reporter RNAs containing a consensus SF2/ASF binding site [29]. We used the CLIP protocol to extend our findings to endogenous cellular mRNAs. CLIP allowed us to sample direct mRNA targets of SF2/ASF under conditions that preserve the in situ specificity of protein-RNA interactions. By coupling CLIP with sub-cellular fractionation we were able to identify mRNAs associated with SF2/ASF in the nucleus, cytoplasm and in the actively translating pool of ribosomes PLoS ONE | www.plosone.org 5 October 2008 | Volume 3 | Issue 10 | e3369