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

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

Author information

1
Center for RNA Biology and Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642.
2
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.

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.

KEYWORDS:

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

PMID:
25422459
PMCID:
PMC4267390
DOI:
10.1073/pnas.1412743111
[Indexed for MEDLINE]
Free PMC Article

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