Fouraux Michael A, Bouvet Philippe, Verkaart Sjoerd, van Venrooij Walther J, Pruijn Ger J M
University of Nijmegen, Nijmegen Center of Molecular Life Sciences, Department of Biochemistry, The Netherlands.
J Mol Biol. 2002 Jul 12;320(3):475-88. doi: 10.1016/s0022-2836(02)00518-1.
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.
Ro核糖核蛋白（Ro RNPs）是进化上保守的小型细胞质RNA-蛋白质复合物，其功能未知。在人类细胞中，Ro RNPs由四种hY RNA之一以及两种核心蛋白组成：Ro60和La。最近，已有文献报道了异质性核糖核蛋白I（hnRNP I）和异质性核糖核蛋白K（hnRNP K）与含有hY1和hY3 RNA的颗粒的关联。另外三种蛋白质，即钙网蛋白、Ro52和RoBP1与Ro RNPs（的亚群）的关联仍存在争议。为了更深入了解Ro RNPs的组成和功能，我们使用针对Ro60和La的单克隆抗体从HeLa细胞提取物中免疫纯化了这些颗粒。通过这种方法，我们鉴定出RNA结合蛋白核仁素是含有hY1或hY3 RNA而非hY4和hY5 RNA的Ro RNP颗粒的一种新亚基。使用体外hY RNA结合试验，我们确定hY1和hY3 RNA富含嘧啶的内部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所有四个RNP基序的存在，但不依赖于C末端RGG框。此外，我们证明，与核仁素和hnRNP K不同，核仁素和hnRNP I可以同时结合到hY1 RNA富含嘧啶的内部环上。我们推测核仁素在hY1和hY3核糖核蛋白通过核仁的生物合成和/或运输以及随后向细胞质的转运中发挥作用。
