Alternative conformations at the RNA-binding surface of the N-terminal U2AF65 RNA recognition motif

The RNA recognition motif (RRM) is one of the most abundant types of eukaryotic RNA binding domains, as exemplified by more than ~600 nonredundant human proteins documented by Pfam1. To date, more than 40 different structures of RRM-containing proteins have been determined, including at least ten distinct complexes with RNA. These structures establish an ~80 residue core composed of four β-strands packed against two α-helices. Two ribonucleoprotein consensus sequences on the central β-strands (β3 and β1, respectively called RNP1 and RNP2) display aromatic and basic residues that are important for RNA affinity. The β-sheet surface and additional N- or C-terminal extensions or loops of the RRM interact with the RNA strand. In many cases, the presence of several RRMs within a given polypeptide contributes additional sequence-specificity. Despite these established themes of RNA recognition by RRM-containing proteins, RRM/RNA specificity is far from predictable, and efforts are ongoing to establish the structural and energetic basis of this important type of RNA recognition. Few high resolution (<1.5Å resolution) X-ray structures of RRM-containing proteins are available. Thus, little is known concerning the roles of alternative conformational states and hydration by bound water molecules during RRM/RNA recognition. In a singular example, the structure of a fragment of hnRNP A1 containing two RRMs was determined at 1.1Å resolution in the absence of nucleic acid2. Spatially-correlated alternative conformations for three residues in the RNP motifs of the N-terminal RRM of hnRNP A1 were observed in the absence of nucleic acid. When compared with the 2.1Å resolution structure bound to DNA ligand3, the presence of the nucleic acid sterically precludes one set of these alternative conformations. Without additional high resolution structural examples of RRMs, it is difficult to conclude whether selective ordering of pre-existing alternative conformations by RNA binding represents a recurring theme of nucleic acid recognition by RRM-containing proteins. The importance of specific RNA interactions during RNA processing is illustrated by the substantial number of human genetic diseases associated with errors in pre-mRNA splicing4. During pre-mRNA splicing, the essential splicing factor U2AF65 recognizes the Py-tract consensus sequence near the 3′ splice site of the pre-mRNA. U2AF65 subsequently facilitates stable association of the pre-mRNA with the U2 small nuclear ribonucleoprotein particle (snRNP), a core component of the spliceosome5; 6. As for many proteins involved in RNA processing, two consecutive RRMs (RRM1 and RRM2) guide Py-tract recognition by U2AF657; 8. In addition to recognizing the Py-tract, the N-terminal U2AF65-RRM1 interacts with U2AF Associated Protein 56KD (UAP56), an RNP unwindase that is required for stable association of the U2 snRNP with the pre-mRNA9. Nuclear magnetic resonance (NMR) structures of the individual U2AF65 RRMs reveal the folds of these domains in the absence of nucleic acid10. Recently, the X-ray structure of a U2AF65 fragment in complex with polyuridine, and an accompanying analysis of site-directed mutant proteins, established the importance of RRM1 residues for polyuridine recognition11. The ability of a Py-tract to direct splicing of a nearby pre-mRNA splice site is proportional to the number of consecutive uridines in the sequence12; 13. However, the Py-tracts of higher eukaryotic pre-mRNAs frequently are interrupted by cytosines, adenosines, or guanosines (50% Uri, 30% Cyt, 10% Ade, 10% Gua)14. Thus, U2AF65 is faced with the problem of specifying Py-tracts to ensure accurate splicing, yet tolerating a variety of natural Py-tract sequences15. The structure of the U2AF65 fragment/polyuridine complex suggests that flexible side chains and bound water molecules may rearrange during recognition of degenerate Py-tract sequences. Here, the X-ray structure of U2AF65-RRM1 at 1.47Å resolution provides a detailed view of the conformation and hydration in the absence of RNA, adding to a miniscule database of high resolution RRM structures. This structure suggests that flexible conformations of three side chains and a loop region of RRM1 may contribute to the ability of U2AF65 to tolerate Py-tracts interrupted by purines. In the one other available atomic resolution structure of an RRM domain (hnRNP A1)2, alternative side chain conformations also participate in nucleic acid binding. Thus, the high resolution view of the U2AF65 RRM supports the possibility of a general role for pre-existing alternative conformations in nucleic acid recognition by RRM-containing proteins.