Structure-guided U2AF65 variant improves recognition and splicing of a defective pre-mRNA

Proc Natl Acad Sci U S A. 2014 Dec 9;111(49):17420-5. doi: 10.1073/pnas.1412743111. Epub 2014 Nov 24.


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.

Keywords: RRM; crystal structure; pre-mRNA splicing; protein engineering; protein–RNA complex.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenine / chemistry
  • Alternative Splicing*
  • Binding Sites
  • Crystallography, X-Ray
  • Escherichia coli / metabolism
  • Genetic Variation
  • Guanine / chemistry
  • Humans
  • Molecular Conformation
  • Mutation
  • Nuclear Proteins / chemistry*
  • Nuclear Proteins / genetics*
  • Protein Binding
  • Protein Engineering
  • RNA / chemistry
  • Ribonucleoproteins / chemistry*
  • Ribonucleoproteins / genetics*
  • Splicing Factor U2AF
  • Uracil / chemistry


  • Nuclear Proteins
  • Ribonucleoproteins
  • Splicing Factor U2AF
  • U2AF2 protein, human
  • Uracil
  • Guanine
  • RNA
  • Adenine

Associated data

  • PDB/4TU7
  • PDB/4TU8
  • PDB/4TU9