Abstract

The large subunit of the human U2 small nuclear ribonucleoprotein particle auxiliary factor (hU2AF⁶⁵) is an essential RNA-splicing factor required for the recognition of the polypyrimidine tract immediately upstream of the 3' splice site. In the present study, we determined the solution structures of two hU2AF⁶⁵ fragments, corresponding to the first and second RNA-binding domains (RBD1 and RBD2, respectively), by nuclear magnetic resonance spectroscopy. The tertiary structure of RBD2 is similar to that of typical RNA-binding domains with the 1–1–2–3–2–4 topology. In contrast, the hU2AF⁶⁵ RBD1 structure has unique features: (i) the 1 helix is elongated by one turn toward the C-terminus; (ii) the loop between 1 and 2 (the 1/2 loop) is much longer and has a defined conformation; (iii) the 2 strand is ¹⁸⁸AVQIN¹⁹², which was not predicted by sequence alignments; and (iv) the 2/3 loop is much shorter. Chemical shift perturbation experiments showed that the U2AF-binding RNA fragments interact with the four -strands of RBD2 whereas, in contrast, they interact with 1, 3 and 4, but not with 2 or the 1/2 loop, of RBD1. The characteristic 1–2 structure of the hU2AF⁶⁵ RBD1 may interact with other proteins, such as UAP56.