The U2AF-homology motif (UHM) mediates protein-protein interactions between factors involved in constitutive RNA splicing. Here we report that the splicing factor SPF45 regulates alternative splicing of the apoptosis regulatory gene FAS (also called CD95). The SPF45 UHM is necessary for this activity and binds UHM-ligand motifs (ULMs) present in the 3′ splice site–recognizing factors U2AF65, SF1 and SF3b155. We describe a 2.1-Å crystal structure of SPF45-UHM in complex with a ULM peptide from SF3b155. Features distinct from those of previously described UHM-ULM structures allowed the design of mutations in the SPF45 UHM that selectively impair binding to individual ULMs. Splicing assays using the ULM-selective SPF45 variants demonstrate that individual UHM-ULM interactions are required for FAS splicing regulation by SPF45 in vivo. Our data suggest that networks of UHM-ULM interactions are involved in regulating alternative splicing.
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We thank the staff at the European Synchrotron Radiation Facility and Swiss Light Source for assistance during data collection, V. Rybin (European Molecular Biology Laboratory, EMBL) for ITC measurements, I. Sinning and I. Tews (University of Heidelberg) for ITC measurement time, G. Stier (EMBL) for expression vectors, and B. Simon (EMBL) for help with NMR measurements. M.H. is a fellow of the Peter and Traudl Engelhorn Foundation. This work was supported by the Deutsche Forschungsgemeinschaft (Sa 823/5) and EU grant 3D Repertoire (LSHG-CT-2005-512028).
The authors declare no competing financial interests.
Expression control of SPF54 by western blot with anti-SPF45 (PDF 111 kb)
Isothermal calorimetry titrations of SPF45-UHM with U2AF65-ULM (PDF 117 kb)
In the cocrystals of SPF45-UHM and SF3b155-ULM5, the N terminus of the peptide is involved in lattice formation (PDF 152 kb)
15N T1p data, 1H-15N heteronuclear NOE data and 1H,15N correlation spectra of SPF45 (PDF 318 kb)
Supplementary GST pull-down data (PDF 346 kb)
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Corsini, L., Bonnal, S., Basquin, J. et al. U2AF-homology motif interactions are required for alternative splicing regulation by SPF45. Nat Struct Mol Biol 14, 620–629 (2007). https://doi.org/10.1038/nsmb1260
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