Department of Zoology, University of Cambridge, Cambridge, United Kingdom.
PLoS One. 2010 Oct 27;5(10):e13673. doi: 10.1371/journal.pone.0013673.
Ro ribonucleoprotein particles (Ro RNPs) consist of a non-coding Y RNA bound by Ro60, La and possibly other proteins. The physiological function of Ro RNPs is controversial as divergent functions have been reported for its different constituents. We have recently shown that Y RNAs are essential for the initiation of mammalian chromosomal DNA replication, whereas Ro RNPs are implicated in RNA stability and RNA quality control. Therefore, we investigate here the functional consequences of RNP formation between Ro60, La and nucleolin proteins with hY RNAs for human chromosomal DNA replication.
METHODOLOGY/PRINCIPAL FINDINGS: We first immunoprecipitated Ro60, La and nucleolin together with associated hY RNAs from HeLa cytosolic cell extract, and analysed the protein and RNA compositions of these precipitated RNPs by Western blotting and quantitative RT-PCR. We found that Y RNAs exist in several RNP complexes. One RNP comprises Ro60, La and hY RNA, and a different RNP comprises nucleolin and hY RNA. In addition about 50% of the Y RNAs in the extract are present outside of these two RNPs. Next, we immunodepleted these RNP complexes from the cytosolic extract and tested the ability of the depleted extracts to reconstitute DNA replication in a human cell-free system. We found that depletion of these RNP complexes from the cytosolic extract does not inhibit DNA replication in vitro. Finally, we tested if an excess of recombinant pure Ro or La protein inhibits Y RNA-dependent DNA replication in this cell-free system. We found that Ro60 and La proteins do not inhibit DNA replication in vitro.
CONCLUSIONS/SIGNIFICANCE: We conclude that RNPs containing hY RNAs and Ro60, La or nucleolin are not required for the function of hY RNAs in chromosomal DNA replication in a human cell-free system, which can be mediated by Y RNAs outside of these RNPs. These data suggest that Y RNAs can support different cellular functions depending on associated proteins.
Ro 核糖核蛋白颗粒（Ro RNPs）由非编码 Y RNA 与 Ro60、La 以及可能的其他蛋白结合而成。Ro RNPs 的生理功能存在争议，因为其不同成分具有不同的功能。我们最近发现 Y RNA 对于哺乳动物染色体 DNA 复制的起始至关重要，而 Ro RNPs 则与 RNA 稳定性和 RNA 质量控制有关。因此，我们在此研究了 Ro60、La 和核仁蛋白与 hY RNA 之间形成 RNP 对人类染色体 DNA 复制的功能影响。
方法/主要发现：我们首先从 HeLa 细胞质细胞提取物中免疫沉淀与相关 hY RNA 一起的 Ro60、La 和核仁蛋白，并通过 Western blot 和定量 RT-PCR 分析这些沉淀 RNP 的蛋白和 RNA 组成。我们发现 Y RNA 存在于几种 RNP 复合物中。一种 RNP 包含 Ro60、La 和 hY RNA，另一种 RNP 包含核仁蛋白和 hY RNA。此外，提取物中约 50%的 Y RNA 存在于这两种 RNP 之外。接下来，我们从细胞质提取物中免疫耗竭这些 RNP 复合物，并测试耗竭提取物在体外重新构建 DNA 复制的能力。我们发现，从细胞质提取物中耗竭这些 RNP 复合物不会抑制体外 DNA 复制。最后，我们测试了过量的重组纯 Ro 或 La 蛋白是否会抑制该无细胞系统中 Y RNA 依赖性 DNA 复制。我们发现 Ro60 和 La 蛋白在体外不会抑制 DNA 复制。
结论/意义：我们的结论是，含有 hY RNA、Ro60、La 或核仁蛋白的 RNP 对于 hY RNA 在人类无细胞系统中进行染色体 DNA 复制的功能不是必需的，这可以由这些 RNP 之外的 Y RNA 介导。这些数据表明，Y RNA 可以根据相关蛋白支持不同的细胞功能。
