Solution Conformation and Thermodynamic Characteristics of RNA Binding by the Splicing Factor U2AF65

The U2 auxiliary factor large subunit (U2AF(65)) is an essential pre-mRNA splicing factor for the initial stages of spliceosome assembly. Tandem RNA recognition motifs (RRM)s of U2AF(65) recognize polypyrimidine tract signals adjacent to 3' splice sites. Despite the central importance of U2AF(65) for splice site recognition, the relative arrangement of the U2AF(65) RRMs and the energetic forces driving polypyrimidine tract recognition remain unknown. Here, the solution conformation of the U2AF(65) RNA binding domain determined using small angle x-ray scattering reveals a bilobal shape without apparent inter-domain contacts. The proximity of the N and C termini within the inter-RRM configuration is sufficient to explain the action of U2AF(65) on spliceosome components located both 5' and 3' to its binding site. Isothermal titration calorimetry further demonstrates that an unusually large enthalpy-entropy compensation underlies U2AF(65) recognition of an optimal polyuridine tract. Qualitative similarities were observed between the pairwise distance distribution functions of the U2AF(65) RNA binding domain and those either previously observed for N-terminal RRMs of Py tract-binding protein that lack interdomain contacts or calculated from the high resolution coordinates of a U2AF(65) deletion variant bound to RNA. To further test this model, the shapes and RNA interactions of the wild-type U2AF(65) RNA binding domain were compared with those of U2AF(65) variants containing either Py tract-binding protein linker sequences or a deletion within the inter-RRM linker. Results of these studies suggest inter-RRM conformational plasticity as a possible means for U2AF(65) to universally identify diverse pre-mRNA splice sites.