Solution structures of the first end second RNA-binding domains of human U2 small nuclear ribonucleoprotein particle auxiliary factor (U2AF65)

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 β1-α1-β2-β3-α2-β4 topology. In contrast, the hU2AF65 RBD1 structure has unique features: (i) the α1 helix is elongated by one turn toward the C-terminus; (ii) the loop between α1 and β2 (the α1/β2 loop) is much longer and has a defined conformation; (iii) the β2 strand is 188AVQIN192, which was not predicted by sequence alignments; and (iv) the β2/β3 loop is much shorter. Chemical shift perturbation experiments showed that the U2AF-binding RNA fragments interact with the four β-strands of RBD2 whereas, in contrast, they interact with β1, β3 and β4, but not with β2 or the α1/β2 loop, of RBD1. The characteristic α1-β2 structure of the hU2AF65 RBD1 may interact with other proteins, such as UAP56.