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Zhang, Y.-Y. ; Madl, T. ; Bagdiul, I.* ; Kern, T. ; Kang, H.-S. ; Zou, P. ; Mäusbacher, N.* ; Sieber, S.A.* ; Krämer, A.* ; Sattler, M.

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

Nucleic Acids Res. 41, 1343-1354 (2013)
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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.
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Publication type Article: Journal article
Document type Scientific Article
Keywords Polypyrimidine Tract Recognition ; Angle Scattering Data ; N-15 Nmr Relaxation ; Factor Sf1 ; Protein Recognition ; Rna ; Spectroscopy ; Dynamics ; Macromolecules ; Conservation
ISSN (print) / ISBN 0305-1048
e-ISSN 1362-4962
Quellenangaben Volume: 41, Issue: 2, Pages: 1343-1354 Article Number: , Supplement: ,
Publisher Oxford University Press
Reviewing status Peer reviewed