A Novel Domain Implicated in the Interactions between pre-mRNA Splicing Factors

Abstract

Splicing Factor 1 (SF1) and U2 snRNP auxiliary factor (U2AF65) form an essential protein complex that recognizes the 3′ splice site during the initial stages of pre-mRNA splicing. A ∼100 amino acid domain of SF1 located between an N-terminal region that is necessary and sufficient U2AF65-interactions, and a C-terminal RNA-binding domain. Despite high sequence conservation from yeast to mammals, the structure and function of this SF1 ‘mystery’ domain is currently unknown. Here, we demonstrate that the SF1 ‘mystery’ domain participates in the SF1 / U2AF65 interface by comparing heat capacity changes and chemical shift differences for U2AF65 association with deletion variants of SF1. Heat capacity changes for association of SF1 with the U2AF65 interacting domain (UHM) are significantly greater than those observed for association with a SF1 peptide composed of the minimal U2AF65-interacting region. In contrast, the heat capacity changes for SF1 peptide/U2AF65UHM association closely matched those predicted from the buried surface area of the complex. Given that heat capacity changes often correlate with the amount of surface area buried by complex formation, one possible explanation for this difference was that additional regions of SF1 participate in the U2AF65UHM interface. To investigate this possibility, the HSQC spectra of 15N-labeled U2AF65-UHM in complex with SF1 C-terminal deletion variants were compared. Chemical shift differences imply that residues from conserved ‘mystery’ domain of SF1 participate in the U2AF65UHM interface. The influence of this SF1 domain on affinity and cooperativity of pre-mRNA recognition by the SF1 / U2AF65 is further investigated by calorimetry and fluorescence anisotropy. These studies aid in elucidating the structural and thermodynamic means for 3′ splice site recognition by the essential SF1 and U2AF65 complex.