title: Structure-guided U2AF65 variant improves recognition and splicing of a defective pre-mRNA.
abstract: Purine interruptions of polypyrimidine (Py) tract splice site signals contribute to human genetic diseases. The essential splicing factor U2AF(65) normally recognizes a Py tract consensus sequence preceding the major class of 3' splice sites. We found that neurofibromatosis- or retinitis pigmentosa-causing mutations in the 5' regions of Py tracts severely reduce U2AF(65) affinity. Conversely, we identified a preferred binding site of U2AF(65) for purine substitutions in the 3' regions of Py tracts. Based on a comparison of new U2AF(65) structures bound to either A- or G-containing Py tracts with previously identified pyrimidine-containing structures, we expected to find that a D231V amino acid change in U2AF(65) would specify U over other nucleotides. We found that the crystal structure of the U2AF(65)-D231V variant confirms favorable packing between the engineered valine and a target uracil base. The D231V amino acid change restores U2AF(65) affinity for two mutated splice sites that cause human genetic diseases and successfully promotes splicing of a defective retinitis pigmentosa-causing transcript. We conclude that reduced U2AF(65) binding is a molecular consequence of disease-relevant mutations, and that a structure-guided U2AF(65) variant is capable of manipulating gene expression in eukaryotic cells.
authors.affiliation: Center for RNA Biology and Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642.
authors.forename: Anant A
authors.initials: AA
authors.lastname: Agrawal
authors.affiliation: Center for RNA Biology and Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642.
authors.forename: Krystle J
authors.initials: KJ
authors.lastname: McLaughlin
authors.affiliation: Center for RNA Biology and Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642.
authors.forename: Jermaine L
authors.initials: JL
authors.lastname: Jenkins
authors.affiliation: Center for RNA Biology and Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642 clara_kielkopf@urmc.rochester.edu.
authors.forename: Clara L
authors.initials: CL
authors.lastname: Kielkopf
mesh_terms: D000225:Adenine / Q000737:chemistry; D017398:Alternative Splicing; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D014644:Genetic Variation; D006147:Guanine / Q000737:chemistry; D006801:Humans; D008968:Molecular Conformation; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics*; D011485:Protein Binding; D015202:Protein Engineering; D012313:RNA / Q000737:chemistry; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000235:genetics*; D000072668:Splicing Factor U2AF; D014498:Uracil / Q000737:chemistry
chemical_list: D009687:Nuclear Proteins; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; D014498:Uracil; D006147:Guanine; D012313:RNA; D000225:Adenine
keywords: RRM; crystal structure; pre-mRNA splicing; protein engineering; protein–RNA complex