Structure-guided U2AF^sup 65^ variant improves recognition and splicing of a defective pre-mRNA

• Published in: Proceedings of the National Academy of Sciences - PNAS 2014-12, Vol.111 (49), p.17420
• Main Authors: Agrawal, Anant A, McLaughlin, Krystle J, Jenkins, Jermaine L, Kielkopf, Clara L
• Format: Article
• Language: English
• Subjects: Amino acids; Binding sites; Crystal structure; Eukaryotes; Gene expression; Genetic disorders; Mutation
• ISSN: 0027-8424; 1091-6490

Purine interruptions of polypyrimidine (Py) tract splice site signals contribute to human genetic diseases. The essential splicing factor U2AF^sup 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^sup 65^ affinity. Conversely, we identified a preferred binding site of U2AF^sup 65^ for purine substitutions in the 3' regions of Py tracts. Based on a comparison of new U2AF^sup 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^sup 65^ would specify U over other nucleotides. We found that the crystal structure of the U2AF^sup 65^-D231V variant confirms favorable packing between the engineered valine and a target uracil base. The D231V amino acid change restores U2AF^sup 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^sup 65^ binding is a molecular consequence of disease-relevant mutations, and that a structure-guided U2AF^sup 65^ variant is capable of manipulating gene expression in eukaryotic cells.