Nucleolin associates with a subset of the human Ro ribonucleoprotein complexes
Nucleolin associates with a subset of the human Ro ribonucleoprotein complexes
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DOI：
10.1016/s0022-2836(02)00518-1
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发表时间：
2002-07-12
影响因子：
5.6
通讯作者：
Pruijn, GJM
中科院分区：
文献类型：
--
作者：
Fouraux, MA;Bouvet, P;Pruijn, GJM
Ro RNP 是进化上保守的、功能未知的小型细胞质 RNA-蛋白质复合物。在人类细胞中，Ro RNP 由四种 hY RNA 之一和两种核心蛋白：Ro60 和 La 组成。最近，已经描述了 hnRNP I 和 hnRNP K 与含有 hY1 和 hY3 RNA 的颗粒的关联。其他三种蛋白质（即钙网蛋白、Ro52 和 RoBPI）与 Ro RNP（子集）的关联仍然存在争议。为了更深入地了解 Ro RNP 的组成和功能，我们使用针对 Ro60 和 La 的单克隆抗体对 HeLa 细胞提取物中的这些颗粒进行了免疫纯化。使用这种方法，我们将 RNA 结合蛋白核仁素鉴定为 Ro RNP 颗粒的新亚基，其中包含 hY1 或 hY3 RNA，但不包含 hY4 和 hY5 RNA。使用体外 hY RNA 结合测定，我们确定 hY1 和 hY3 RNA 的内部富含嘧啶的环对于核仁素的结合至关重要。这种结合严重依赖于核仁素的所有四个 RNP 基序的存在，但不依赖于 C 端 RGG-box 的存在。此外，我们证明，与核仁素和 hnRNP K 不同，核仁素和 hnRNP I 可以同时结合 hY1 RNA 内部富含嘧啶的环。我们假设核仁素在 hY1 和 hY3 RNP 的生物发生和/或运输中发挥作用，通过核仁并随后转运到细胞质。 (C) 2002 Elsevier Science Ltd. 保留所有权利。
Ro RNPs are evolutionarily conserved, small cytoplasmic RNA-protein complexes with an unknown function. In human cells, Ro RNPs consist of one of the four hY RNAs and two core proteins: Ro60 and La. Recently, the association of hnRNP I and hnRNP K with particles containing hY1 and hY3 RNAs has been described. The association of three other proteins, namely calreticulin, Ro52 and RoBPI, with (subsets of) the Ro RNPs is still controversial. To gain more insight into the composition and function of the Ro RNPs, we have immunopurified these particles from HeLa cell extracts using monoclonal antibodies against Ro60 and La. Using this approach, we have identified the RNA-binding protein nucleolin as a novel subunit of Ro RNP particles containing hY1 or hY3 RNA, but not hY4 and hY5 RNA. Using an in vitro hY RNA-binding assay we established that the internal pyrimidine-rich loop of hY1 and hY3 RNA is essential for the association of nucleolin. The binding is critically dependent on the presence of all four RNP motifs of nucleolin, but not of the C-terminal RGG-box. Moreover, we demonstrate that, in contrast to nucleolin and hnRNP K, nucleolin and hnRNP I can bind simultaneously to the internal pyrimidine-rich loop of hY1 RNA. We postulate that nucleolin functions in the biogenesis and/or trafficking of hY1 and hY3 RNPs through the nucleolus and subsequent transport to the cytoplasm. (C) 2002 Elsevier Science Ltd. All rights reserved.