Wan L, Kim J K, Pollard V W, Dreyfuss G
Howard Hughes Medical Institute and Department of Biochemistry & Biophysics, University of Pennsylvania School of Medicine, Philadelphia 19104-6148, USA.
J Biol Chem. 2001 Mar 9;276(10):7681-8. doi: 10.1074/jbc.M010207200. Epub 2000 Dec 11.
The heterogeneous nuclear ribonucleoprotein (hn- RNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of approximately 80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.
异质性核核糖核蛋白（hn-RNP）C蛋白是真核细胞核中最丰富的前体mRNA结合蛋白之一，具有单个核糖核蛋白基序RNA结合结构域。RNA结合结构域（RBD）由大约80 - 100个氨基酸组成，其结构已被确定。然而，关于RBD中特定氨基酸在与RNA结合中的作用，人们了解得相对较少。我们设计了一种基于噬菌体展示的筛选方法，用于快速鉴定hnRNP C1中对其与RNA结合至关重要的氨基酸。对鉴定出的突变体进一步测试其与聚（U）-琼脂糖的结合，聚（U）-琼脂糖是野生型hnRNP C1以高亲和力结合的底物。我们发现RBD中既有先前预测的高度保守残基，也有其他残基对C1与RNA的结合至关重要。我们还在该蛋白羧基末端附近富含亮氨酸的C1 - C1相互作用结构域中鉴定出三个突变，这些突变既消除了C1的寡聚化，又降低了RNA结合。这些结果表明，虽然RBD是C1与RNA结合的主要决定因素，但C1 - C1相互作用结构域中的残基也影响该蛋白的RNA结合活性。我们描述的实验方法通常应适用于筛选和鉴定在蛋白质与核酸底物结合中起作用的氨基酸。
