SF1 Phosphorylation Enhances Specific Binding to U2AF^sup 65^ and Reduces Binding to 3'-Splice-Site RNA

• Published in: Biophysical journal Vol. 111; no. 12; p. 2570
• Main Authors: Chatrikhi, Rakesh, Wang, Wenhua, Gupta, Ankit, Loerch, Sarah, Maucuer, Alexandre, Kielkopf, Clara L
• Format: Journal Article
• Language: English
• Published: New York Biophysical Society 20-12-2016
• Subjects: Anisotropy; Binding sites; Fluorescence; Kinases; Phosphorylation; Ribonucleic acid; RNA
• ISSN: 0006-3495; 1542-0086

Splicing factor 1 (SF1) recognizes 3' splice sites of the major class of introns as a ternary complex with U2AF^sup 65^ and U2AF^sup 35^ splicing factors. A conserved SPSP motif in a coiled-coil domain of SF1 is highly phosphorylated in proliferating human cells and is required for cell proliferation. The UHM kinase 1 (UHMK1), also called KIS, double-phosphorylates both serines of this SF1 motif. Here, we use isothermal titration calorimetry to demonstrate that UHMK1 phosphorylation of the SF1 SPSP motif slightly enhances specific binding of phospho-SF1 to its cognate U2AF^sup 65^ protein partner. Conversely, quantitative fluorescence anisotropy RNA binding assays and isothermal titration calorimetry experiments establish that double-SPSP phosphorylation reduces phospho-SF1 and phospho-SF1-U2AF^sup 65^ binding affinities for either optimal or suboptimal splice-site RNAs. Domain-substitution and mutagenesis experiments further demonstrate that arginines surrounding the phosphorylated SF1 loop are required for cooperative 3' splice site recognition by the SF1-U2AF^sup 65^ complex (where cooperativity is defined as a nonadditive increase in RNA binding by the protein complex relative to the individual proteins). In the context of local, intracellular concentrations, the subtle effects of SF1 phosphorylation on its associations with U2AF^sup 65^ and splice-site RNAs are likely to influence pre-mRNA splicing. However, considering roles for SF1 in pre-mRNA retention and transcriptional repression, as well as in splicing, future comprehensive investigations are needed to fully explain the requirement for SF1 SPSP phosphorylation in proliferating human cells.