title: Structure of phosphorylated SF1 bound to U2AF⁶⁵ in an essential splicing factor complex.
abstract: The essential splicing factors U2AF⁶⁵ and SF1 cooperatively bind consensus sequences at the 3' end of introns. Phosphorylation of SF1 on a highly conserved "SPSP" motif enhances its interaction with U2AF⁶⁵ and the pre-mRNA. Here, we reveal that phosphorylation induces essential conformational changes in SF1 and in the SF1/U2AF⁶⁵/3' splice site complex. Crystal structures of the phosphorylated (P)SF1 domain bound to the C-terminal domain of U2AF⁶⁵ at 2.29 Å resolution and of the unphosphorylated SF1 domain at 2.48 Å resolution demonstrate that phosphorylation induces a disorder-to-order transition within a previously unknown SF1/U2AF⁶⁵ interface. We find by small-angle X-ray scattering that the local folding of the SPSP motif transduces into global conformational changes in the nearly full-length (P)SF1/U2AF⁶⁵/3' splice site assembly. We further determine that SPSP phosphorylation and the SF1/U2AF⁶⁵ interface are essential in vivo. These results offer a structural prototype for phosphorylation-dependent control of pre-mRNA splicing factors.
authors.affiliation: Center for RNA Biology and Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
authors.forename: Wenhua
authors.initials: W
authors.lastname: Wang
authors.forename: Alexandre
authors.initials: A
authors.lastname: Maucuer
authors.forename: Ankit
authors.initials: A
authors.lastname: Gupta
authors.forename: Valérie
authors.initials: V
authors.lastname: Manceau
authors.forename: Karen R
authors.initials: KR
authors.lastname: Thickman
authors.forename: William J
authors.initials: WJ
authors.lastname: Bauer
authors.forename: Scott D
authors.initials: SD
authors.lastname: Kennedy
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Wedekind
authors.forename: Michael R
authors.initials: MR
authors.lastname: Green
authors.forename: Clara L
authors.initials: CL
authors.lastname: Kielkopf
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D049109:Cell Proliferation; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000502:physiology; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D006860:Hydrogen Bonding; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000737:chemistry* / Q000502:physiology; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D022821:RNA Splice Sites; D000072260:RNA Splicing Factors; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000502:physiology; D000072668:Splicing Factor U2AF; D014157:Transcription Factors / Q000737:chemistry* / Q000502:physiology
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D022821:RNA Splice Sites; D000072260:RNA Splicing Factors; D012261:Ribonucleoproteins; C107467:SF1 protein, human; D000072668:Splicing Factor U2AF; D014157:Transcription Factors; C000607494:U2AF2 protein, human; C000607495:Zrsr1 protein, mouse