Muto Yutaka, Pomeranz Krummel Daniel, Oubridge Chris, Hernandez Helena, Robinson Carol V, Neuhaus David, Nagai Kiyoshi
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
J Mol Biol. 2004 Jul 30;341(1):185-98. doi: 10.1016/j.jmb.2004.04.078.
The spliceosomal U1C protein is critical to the initiation and regulation of precursor messenger RNA (pre-mRNA) splicing, as part of the U1 small nuclear ribonucleoprotein particle (snRNP). We have produced full-length and 61 residue constructs of human U1C in soluble form in Escherichia coli. Atomic absorption spectroscopy and mass spectrometry show that both constructs contain one Zn atom and are monomeric. Gelmobility-shift assays showed that one molecule of recombinant U1C, either full-length or 61 residue construct, can be incorporated into the U1 snRNP core domain in the presence of U1 70k. This result is in perfect agreement with the previous experiment with U1C isolated from the HeLa U1 snRNP showing that the recombinant U1C is functionally active. We have determined the solution structure of the N-terminal 61 residue construct of U1C by NMR. A Cys(2)His(2)-type zinc finger, distinct from the TFIIIA-type, is extended at its C terminus by two additional helices. The two Zn-coordinating histidine residues are separated by a five residue loop. The conserved basic residues in the first two helices and the intervening loop may be involved in RNA binding. The opposite beta-sheet face with two surface-exposed Tyr residues may be involved in protein contacts. Both the full-length and 61 residue constructs of human U1C fail to bind RNA containing the 5' splice site sequence, in contrast to what has been reported for the Saccharomyces cerevisiae orthologue.
作为U1小核核糖核蛋白颗粒（snRNP）的一部分，剪接体U1C蛋白对于前体信使RNA（pre-mRNA）剪接的起始和调控至关重要。我们已在大肠杆菌中以可溶形式制备了人U1C的全长和61个残基的构建体。原子吸收光谱法和质谱分析表明，这两种构建体均含有一个锌原子且为单体。凝胶迁移率变动分析表明，在存在U1 70k的情况下，一分子重组U1C（全长或61个残基的构建体）可被整合到U1 snRNP核心结构域中。这一结果与先前从HeLa U1 snRNP中分离出U1C的实验完全一致，表明重组U1C具有功能活性。我们已通过核磁共振确定了U1C的N端61个残基构建体的溶液结构。一种不同于TFIIIA型的Cys(2)His(2)型锌指在其C端延伸出另外两个螺旋。两个与锌配位的组氨酸残基被一个五个残基的环隔开。前两个螺旋和中间环中的保守碱性残基可能参与RNA结合。具有两个表面暴露的酪氨酸残基的相对β-折叠面可能参与蛋白质相互作用。与酿酒酵母同源物的报道相反，人U1C的全长和61个残基构建体均未能结合含有5'剪接位点序列的RNA。
