Structure, phosphorylation and U2AF65 binding of the N-terminal domain of splicing factor 1 during 3'-splice site recognition

• Published in: Nucleic acids research 2013-01, Vol.41 (2), p.1343-1354
• Main Authors: Zhang, Yun, Madl, Tobias, Bagdiul, Ivona, Kern, Thomas, Kang, Hyun-Seo, Zou, Peijian, Mäusbacher, Nina, Sieber, Stephan A, Krämer, Angela, Sattler, Michael
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Conformation; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - metabolism; Homology; Humans; Hydrophobicity; Ionizing radiation; Models, Molecular; Molecular Sequence Data; Nuclear Proteins - chemistry; Nuclear Proteins - metabolism; Phosphorylation; Protein Binding; Protein Interaction Domains and Motifs; Protein Structure, Secondary; Ribonucleoproteins (U2 small nuclear); Ribonucleoproteins - chemistry; Ribonucleoproteins - metabolism; RNA; RNA - metabolism; RNA Splice Sites; RNA Splicing Factors; Sequence Homology, Amino Acid; Serine; Serine - metabolism; Spliceosomes; Splicing Factor U2AF; splicing factors; Structural Biology; Transcription Factors - chemistry; Transcription Factors - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gks1097

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.