Caceres1* Abstract Identification of Nuclear and Cytoplasmic mRNA Targets for the Shuttling Protein SF2/ASF Jeremy R. Sanford1,2¤*, Pedro Coutinho1, Jamie A. Hackett1, Xin Wang 2 , William Ranahan2, Javier F. 1 MRC Human Genetics Unit, Western General Hospital, Edinburgh, United Kingdom, 2 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America The serine and arginine-rich protein family (SR proteins) are highly conserved regulators of pre-mRNA splicing. SF2/ASF, a prototype member of the SR protein family, is a multifunctional RNA binding protein with roles in pre-mRNA splicing, mRNA export and mRNA translation. These observations suggest the intriguing hypothesis that SF2/ASF may couple splicing and translation of specific mRNA targets in vivo. Unfortunately the paucity of endogenous mRNA targets for SF2/ASF has hindered testing of this hypothesis. Here, we identify endogenous mRNAs directly cross-linked to SF2/ASF in different sub- cellular compartments. Cross-Linking Immunoprecipitation (CLIP) captures the in situ specificity of protein-RNA interaction and allows for the simultaneous identification of endogenous RNA targets as well as the locations of binding sites within the RNA transcript. Using the CLIP method we identified 326 binding sites for SF2/ASF in RNA transcripts from 180 protein coding genes. A purine-rich consensus motif was identified in binding sites located within exon sequences but not introns. Furthermore, 72 binding sites were occupied by SF2/ASF in different sub-cellular fractions suggesting that these binding sites may influence the splicing or translational control of endogenous mRNA targets. We demonstrate that ectopic expression of SF2/ASF regulates the splicing and polysome association of transcripts derived from the SFRS1, PABC1, NETO2 and ENSA genes. Taken together the data presented here indicate that SF2/ASF has the capacity to co-regulate the nuclear and cytoplasmic processing of specific mRNAs and provide further evidence that the nuclear history of an mRNA may influence its cytoplasmic fate. Citation: Sanford JR, Coutinho P, Hackett JA, Wang X, Ranahan W, et al. (2008) Identification of Nuclear and Cytoplasmic mRNA Targets for the Shuttling Protein SF2/ASF. PLoS ONE 3(10): e3369. doi:10.1371/journal.pone.0003369 Editor: Stefan Maas, Lehigh University, United States of America Received July 26, 2008; Accepted July 31, 2008; Published October 8, 2008 Copyright: 2008 Sanford et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by the Medical Research Council (J.F.C., P.C. and J.A.H) plus additional funds from EURASNET (European Alternative splicing Network) to J.F.C.. J.R.S. was initially supported by a postdoctoral fellowship from the Caledonian Research Foundation and is currently funded by American Heart Association Scientist Development Grant #0830206N. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sanford@biology.ucsc.edu (JRS); Javier.Caceres@hgu.mrc.ac.uk (JFC) ¤ Current address: Molecular, Cellular and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, United States of America Introduction Eukaryotic messenger RNA (mRNA) must be processed prior to programming protein synthesis. The minimal modifications for most mRNAs include capping, pre-mRNA splicing and polyad- enylation [1]. These reactions occur in the nucleus and must be completed prior to nuclear export of the mRNA to the cytoplasm. The cytoplasmic fate of mRNA is also subject to regulation at the level of localization, stability and translational efficiency [2]. RNA processing reactions have been extensively studied using biochem- ical systems; however, these are functionally linked in living cells providing increased efficiency and regulatory potential for gene expression [3]. The molecular mechanisms responsible for the coupling of post-transcriptional regulatory networks are poorly understood. A subset of multi-functional mRNA binding proteins operating at the interface of distinct RNA processing machineries may contribute to coupling of post-transcriptional gene expression. The Serine and Arginine-rich (SR) protein family consists of eight phylogenetically conserved proteins ranging in molecular weight from 20–75 kDa. SR proteins have well-characterized roles in pre- mRNA splicing including exon definition and assembly of the mature spliceosome [4,5]. A subset of the SR protein family also function outside pre-mRNA splicing [6]. Indeed, we recently established a role of the shuttling SR protein SF2/ASF in mRNA translation [7,8]. SF2/ASF enhances translation initiation by promoting phosphorylation of the translational repressor protein 4E-BP1 by the mammalian target of rapamycin (mTOR) [9]. Further roles for SRp20 and 9G8 in internal ribosome entry site (IRES)-mediated translation and intronless mRNA export have been documented [10,11]. These observations suggest the hypothesis that shuttling SR proteins may couple nuclear and cytoplasmic steps of pre-mRNA processing. Elucidating endogenous mRNA targets that are regulated by SF2/ASF in both the nucleus and cytoplasm is critical to testing the validity of this hypothesis. The cross-linking immunoprecipitation (CLIP) method is a powerful method for elucidating the target specificity of RNA binding proteins in vivo [12]. In this protocol, living cells are exposed to UV irradiation to induce covalent cross-links between RNA binding proteins and their in situ RNA targets. Prior to immunoprecipitation of a specific RNA binding protein lysates are treated with RNase in order to generate 40–60 nt cross-linked RNA tags. Co-purifying RNA tags are then cloned and sequenced PLoS ONE | www.plosone.org 1 October 2008 | Volume 3 | Issue 10 | e3369