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: Anant A Agrawal, Krystle J McLaughlin, Jermaine L Jenkins, Clara L Kielkopf
• Journal: Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 111 Issue 49 Pg. 17420-5 (Dec 09 2014) ISSN: 1091-6490 [Electronic] United States
• PMID: 25422459 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

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

Topics

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