Thisted T, Lyakhov D L, Liebhaber S A
Howard Hughes Medical Institute and the Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 2001 May 18;276(20):17484-96. doi: 10.1074/jbc.M010594200. Epub 2001 Feb 2.
The KH domain mediates RNA binding in a wide range of proteins. Here we investigate the RNA-binding properties of two abundant RNA-binding proteins, alphaCP-2KL and heterogeneous nuclear ribonucleoprotein (hnRNP) K. These proteins constitute the major poly(C) binding activity in mammalian cells, are closely related on the basis of the structures and positioning of their respective triplicated KH domains, and have been implicated in a variety of post-transcriptional controls. By using SELEX, we have obtained sets of high affinity RNA targets for both proteins. The primary and secondary structures necessary for optimal protein binding were inferred in each case from SELEX RNA sequence comparisons and confirmed by mutagenesis and structural mapping. The target sites for alphaCP-2KL and hnRNP K were both enriched for cytosine bases and were presented in a single-stranded conformation. In contrast to these shared characteristics, the optimal target sequence for hnRNP K is composed of a single short "C-patch" compatible with recognition by a single KH domain whereas that for alphaCP-2KL encompassed three such C-patches suggesting more extensive interactions. The binding specificities of the respective SELEX RNAs were confirmed by testing their interactions with native proteins in cell extracts, and the importance of the secondary structure in establishing an optimized alphaCP-2KL-binding site was supported by comparison of SELEX target structure with that of the native human alpha-globin 3'-untranslated region. These data indicate that modes of macromolecular interactions of arrayed KH domains can differ even among closely related KH proteins and that binding affinities are substantially dependent on the presentation of the target site within the RNA secondary structure.
KH 结构域介导多种蛋白质与 RNA 的结合。在此，我们研究了两种丰富的 RNA 结合蛋白，即 αCP-2KL 和异质性核核糖核蛋白（hnRNP）K 的 RNA 结合特性。这些蛋白构成了哺乳动物细胞中主要的聚（C）结合活性，基于其各自三重 KH 结构域的结构和定位密切相关，并参与了多种转录后调控。通过使用 SELEX，我们获得了这两种蛋白的高亲和力 RNA 靶标集。在每种情况下，通过 SELEX RNA 序列比较推断出最佳蛋白结合所需的一级和二级结构，并通过诱变和结构图谱进行了确认。αCP-2KL 和 hnRNP K 的靶位点均富含胞嘧啶碱基，并呈单链构象。与这些共同特征相反，hnRNP K 的最佳靶序列由与单个 KH 结构域识别兼容的单个短“C 补丁”组成，而 αCP-2KL 的最佳靶序列包含三个这样的 C 补丁，表明相互作用更广泛。通过测试它们与细胞提取物中天然蛋白的相互作用，证实了各自 SELEX RNA 的结合特异性，并且通过将 SELEX 靶标结构与天然人α-珠蛋白 3'非翻译区的结构进行比较，支持了二级结构在建立优化的αCP-2KL 结合位点中的重要性。这些数据表明，即使在密切相关的 KH 蛋白中，排列的 KH 结构域的大分子相互作用模式也可能不同，并且结合亲和力在很大程度上取决于 RNA 二级结构中靶位点的呈现。
