title: Alternative conformations at the RNA-binding surface of the N-terminal U2AF(65) RNA recognition motif.
abstract: The essential pre-mRNA splicing factor, U2 auxiliary factor 65KD (U2AF(65)) recognizes the polypyrimidine tract (Py-tract) consensus sequence of the pre-mRNA using two RNA recognition motifs (RRMs), the most prevalent class of eukaryotic RNA-binding domain. The Py-tracts of higher eukaryotic pre-mRNAs are often interrupted with purines, yet U2AF(65) must identify these degenerate Py-tracts for accurate pre-mRNA splicing. Previously, the structure of a U2AF(65) variant in complex with poly(U) RNA suggested that rearrangement of flexible side-chains or bound water molecules may contribute to degenerate Py-tract recognition by U2AF(65). Here, the X-ray structure of the N-terminal RRM domain of U2AF(65) (RRM1) is described at 1.47 A resolution in the absence of RNA. Notably, RNA-binding by U2AF(65) selectively stabilizes pre-existing alternative conformations of three side-chains located at the RNA interface (Arg150, Lys225, and Arg227). Additionally, a flexible loop connecting the beta2/beta3 strands undergoes a conformational change to interact with the RNA. These pre-existing alternative conformations may contribute to the ability of U2AF(65) to recognize a variety of Py-tract sequences. This rare, high-resolution view of an important member of the RRM class of RNA-binding domains highlights the role of alternative side-chain conformations in RNA recognition.
authors.affiliation: Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205, USA.
authors.forename: Karen R
authors.initials: KR
authors.lastname: Thickman
authors.forename: E Allen
authors.initials: EA
authors.lastname: Sickmier
authors.forename: Clara L
authors.initials: CL
authors.lastname: Kielkopf
mesh_terms: D020816:Amino Acid Motifs; D001059:Apoproteins / Q000737:chemistry; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012322:RNA Precursors / Q000737:chemistry; D022821:RNA Splice Sites; D016601:RNA-Binding Proteins / Q000737:chemistry*; D012261:Ribonucleoproteins / Q000737:chemistry*; D000072668:Splicing Factor U2AF; D013329:Structure-Activity Relationship; D014867:Water / Q000378:metabolism
chemical_list: D001059:Apoproteins; D009687:Nuclear Proteins; D012322:RNA Precursors; D022821:RNA Splice Sites; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; D014867:Water