Structure of Phosphorylated SF1 Bound to U2AF65 in an Essential Splicing Factor Complex

The essential splicing factors U2AF(65) 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 U2AF(65) and the pre-mRNA. Here, we reveal that phosphorylation induces essential conformational changes in SF1 and in the SF1/U2AF(65)/3' splice site complex. Crystal structures of the phosphorylated (P)SF1 domain bound to the C-terminal domain of U2AF(65) at 2.29 angstrom resolution and of the unphosphorylated SF1 domain at 2.48 angstrom resolution demonstrate that phosphorylation induces a disorder-to-order transition within a previously unknown SF1/U2AF(65) 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/U2AF(65)/3' splice site assembly. We further determine that SPSP phosphorylation and the SF1/U2AF(65) interface are essential in vivo. These results offer a structural prototype for phosphorylation-dependent control of pre-mRNA splicing factors.