title: Structure, phosphorylation and U2AF65 binding of the N-terminal domain of splicing factor 1 during 3'-splice site recognition.
abstract: Recognition of the 3'-splice site is a key step in pre-mRNA splicing and accomplished by a dynamic complex comprising splicing factor 1 (SF1) and the U2 snRNP auxiliary factor 65-kDa subunit (U2AF65). Both proteins mediate protein-protein and protein-RNA interactions for cooperative RNA-binding during spliceosome assembly. Here, we report the solution structure of a novel helix-hairpin domain in the N-terminal region of SF1 (SF1(NTD)). The nuclear magnetic resonance- and small-angle X-ray scattering-derived structure of a complex of the SF1(NTD) with the C-terminal U2AF homology motif domain of U2AF65 (U2AF65(UHM)) reveals that, in addition to the known U2AF65(UHM)-SF1 interaction, the helix-hairpin domain forms a secondary, hydrophobic interface with U2AF65(UHM), which locks the orientation of the two subunits. Mutational analysis shows that the helix hairpin is essential for cooperative formation of the ternary SF1-U2AF65-RNA complex. We further show that tandem serine phosphorylation of a conserved Ser80-Pro81-Ser82-Pro83 motif rigidifies a long unstructured linker in the SF1 helix hairpin. Phosphorylation does not significantly alter the overall conformations of SF1, SF1-U2AF65 or the SF1-U2AF65-RNA complexes, but slightly enhances RNA binding. Our results indicate that the helix-hairpin domain of SF1 is required for cooperative 3'-splice site recognition presumably by stabilizing a unique quaternary arrangement of the SF1-U2AF65-RNA complex.
authors.affiliation: Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany.
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Tobias
authors.initials: T
authors.lastname: Madl
authors.forename: Ivona
authors.initials: I
authors.lastname: Bagdiul
authors.forename: Thomas
authors.initials: T
authors.lastname: Kern
authors.forename: Hyun-Seo
authors.initials: HS
authors.lastname: Kang
authors.forename: Peijian
authors.initials: P
authors.lastname: Zou
authors.forename: Nina
authors.initials: N
authors.lastname: Mäusbacher
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Sieber
authors.forename: Angela
authors.initials: A
authors.lastname: Krämer
authors.forename: Michael
authors.initials: M
authors.lastname: Sattler
mesh_terms: D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D012313:RNA / Q000378:metabolism; D022821:RNA Splice Sites; D000072260:RNA Splicing Factors; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D000072668:Splicing Factor U2AF; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
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; D012694:Serine; D012313:RNA