SF1 Phosphorylation Enhances Specific Binding to U2AF65 and Reduces Binding to 3'-Splice-Site RNA.

摘要: Splicing factor 1 (SF1) recognizes 3' splice sites of the major class introns as a ternary complex with U2AF65 and U2AF35 splicing factors. A conserved SPSP motif in coiled-coil domain SF1 is highly phosphorylated proliferating human cells required for cell proliferation. The UHM kinase (UHMK1), also called KIS, double-phosphorylates both serines this motif. Here, we use isothermal titration calorimetry to demonstrate that UHMK1 phosphorylation slightly enhances specific binding phospho-SF1 its cognate protein partner. Conversely, quantitative fluorescence anisotropy RNA assays experiments establish double-SPSP reduces phospho-SF1-U2AF65 affinities either optimal or suboptimal splice-site RNAs. Domain-substitution mutagenesis further arginines surrounding loop are cooperative site recognition by SF1-U2AF65 (where cooperativity defined nonadditive increase relative individual proteins). In context local, intracellular concentrations, subtle effects on associations RNAs likely influence pre-mRNA splicing. However, considering roles retention transcriptional repression, well splicing, future comprehensive investigations needed fully explain requirement cells.