Görlach M, Burd C G, Dreyfuss G
Howard Hughes Medical Institute, Philadelphia, Pennsylvania.
J Biol Chem. 1994 Sep 16;269(37):23074-8.
The hnRNP C proteins (C1/C2) are tenacious nuclear pre-mRNA-binding proteins that belong to the large RNP motif family of RNA-binding proteins. This motif identifies an RNA-binding domain (RBD) that consists of a four-stranded antiparallel beta-sheet packed against two alpha-helices. Despite considerable information on the structure of the hnRNP C RBD, little is known about its RNA-binding properties. To address this we used in vitro selection/amplification from pools of random sequence RNA to determine the RNA-binding specificity of hnRNP C1. After 8 rounds of selection/amplification nearly all RNAs contained contiguous stretches of at least 5 U residues, and filter-binding assays demonstrated that this sequence constitutes a high-affinity (Kd = 170 nM) binding site for hnRNP C1. The highest affinity we measured for hnRNP C1 was for r(U)14 (Kd = 14 nM). An RBD-containing peptide fragment of hnRNP C1 (amino acids 2-94) bound oligoribonucleotides containing an hnRNP C1 high-affinity binding site with nearly equal affinity to that of hnRNP C1. Unlike hnRNP C1, however, this peptide also bound oligoribonucleotides that do not contain high-affinity hnRNP C1-binding sites. We identified a region of 10 amino acids, immediately COOH-terminal to the RNP motif (amino acids 95-104), that prevents the minimal RBD from binding nonspecific RNA ligands. We propose that the highly conserved beta alpha beta beta alpha beta core structure of the RNP motif RBD confers a general RNA binding activity to RNP motif RBDs and that the determinants of RNA-binding specificity reside in the most variable regions, the loops connecting the beta-strands and/or the contiguous NH2 and COOH termini of the RBD.
异质性核糖核蛋白C蛋白（C1/C2）是一类顽固的核内前体mRNA结合蛋白，属于RNA结合蛋白的大RNP基序家族。该基序识别一个RNA结合结构域（RBD），其由一个四链反平行β折叠堆积在两个α螺旋上组成。尽管关于异质性核糖核蛋白C的RBD结构已有大量信息，但对其RNA结合特性却知之甚少。为了解决这个问题，我们从随机序列RNA库中进行体外筛选/扩增，以确定异质性核糖核蛋白C1的RNA结合特异性。经过8轮筛选/扩增后，几乎所有RNA都含有至少5个连续的U残基片段，滤膜结合试验表明该序列构成了异质性核糖核蛋白C1的高亲和力（Kd = 170 nM）结合位点。我们测得异质性核糖核蛋白C1的最高亲和力是针对r(U)14（Kd = 14 nM）。异质性核糖核蛋白C1的一个含RBD的肽片段（氨基酸2 - 94）与含有异质性核糖核蛋白C1高亲和力结合位点的寡核糖核苷酸结合，其亲和力与异质性核糖核蛋白C1几乎相等。然而，与异质性核糖核蛋白C1不同的是，该肽也能结合不含有异质性核糖核蛋白C1高亲和力结合位点的寡核糖核苷酸。我们鉴定出一个位于RNP基序紧邻COOH端的10个氨基酸区域（氨基酸95 - 104），该区域可阻止最小RBD与非特异性RNA配体结合。我们推测，RNP基序RBD的高度保守的β-α-β-β-α-β核心结构赋予RNP基序RBD一般的RNA结合活性，而RNA结合特异性的决定因素存在于最可变的区域，即连接β链的环和/或RBD相邻的NH2和COOH末端。
