A Broad Range of Conformations Contribute to the Solution Ensemble of the Essential Splicing Factor U2AF⁶⁵

U2AF⁶⁵ is essential for pre-mRNA splicing in most eukaryotes. Two consecutive RNA recognition motifs (RRM) of U2AF⁶⁵ recognize a polypyrimidine tract at the 3′ splice site. Here, we use small-angle X-ray scattering to demonstrate that the tandem U2AF⁶⁵ RRMs exhibit a broad range of conformations in the solution ensemble. The majority of U2AF⁶⁵ conformations exhibit few contacts between the RRMs, such as observed in the crystal structure. A subpopulation adopts tight inter-RRM contacts, such as independently reported based on paramagnetic relaxation enhancements. These complementary structural methods demonstrate that diverse splice sites have the opportunity to select compact or extended inter-RRM proximities from the U2AF⁶⁵ conformational pool