title: Conserved proline-directed phosphorylation regulates SR protein conformation and splicing function.
abstract: The alternative splicing of human genes is dependent on SR proteins, a family of essential splicing factors whose name derives from a signature C-terminal domain rich in arginine-serine dipeptide repeats (RS domains). Although the SRPKs (SR-specific protein kinases) phosphorylate these repeats, RS domains also contain prolines with flanking serines that are phosphorylated by a second family of protein kinases known as the CLKs (Cdc2-like kinases). The role of specific serine-proline phosphorylation within the RS domain has been difficult to assign since CLKs also phosphorylate arginine-serine dipeptides and, thus, display overlapping residue specificities with the SRPKs. In the present study, we address the effects of discrete serine-proline phosphorylation on the conformation and cellular function of the SR protein SRSF1 (SR protein splicing factor 1). Using chemical tagging and dephosphorylation experiments, we show that modification of serine-proline dipeptides broadly amplifies the conformational ensemble of SRSF1. The induction of these new structural forms triggers SRSF1 mobilization in the nucleus and alters its binding mechanism to an exonic splicing enhancer in precursor mRNA. These physical events correlate with changes in the alternative splicing of over 100 human genes based on a global splicing assay. Overall, these studies draw a direct causal relationship between a specific type of chemical modification in an SR protein and the regulation of alternative gene splicing programmes.
authors.affiliation: *Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0636, U.S.A.
authors.forename: Malik M
authors.initials: MM
authors.lastname: Keshwani
authors.affiliation: *Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0636, U.S.A.
authors.forename: Brandon E
authors.initials: BE
authors.lastname: Aubol
authors.affiliation: *Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0636, U.S.A.
authors.forename: Laurent
authors.initials: L
authors.lastname: Fattet
authors.affiliation: *Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0636, U.S.A.
authors.forename: Chen-Ting
authors.initials: CT
authors.lastname: Ma
authors.affiliation: †Department of Cellular & Molecular Medicine, University of California, San Diego, La Jolla, CA 92093-0636, U.S.A.
authors.forename: Jinsong
authors.initials: J
authors.lastname: Qiu
authors.affiliation: ‡Department of Chemistry & Biochemistry, University of California, San Diego, La Jolla, CA 92093-0636, U.S.A.
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Jennings
authors.affiliation: †Department of Cellular & Molecular Medicine, University of California, San Diego, La Jolla, CA 92093-0636, U.S.A.
authors.forename: Xiang-Dong
authors.initials: XD
authors.lastname: Fu
authors.affiliation: *Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0636, U.S.A.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Adams
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism; D017124:Conserved Sequence; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011392:Proline / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D013379:Substrate Specificity
chemical_list: D009687:Nuclear Proteins; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000068103:Serine-Arginine Splicing Factors; D012694:Serine; D011392:Proline; C066780:Clk dual-specificity kinases; C087728:SRPK1 protein, human; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases