RNA structure and RNA-protein interactions in purified yeast U6 snRNPs
RNA structure and RNA-protein interactions in purified yeast U6 snRNPs
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DOI：
10.1016/j.jmb.2005.12.013
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发表时间：
2006-03-10
影响因子：
5.6
通讯作者：
Lührmann, R
中科院分区：
文献类型：
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作者：
Karaduman, R;Fabrizio, P;Lührmann, R
U6小核RNA （snRNA）在剪接体的组装和剪接的催化过程中发生了主要的构象变化。它与特定的Prp24p蛋白和一组7个LSm2p-8p蛋白结合，形成U6小核核糖核蛋白（snRNP）。这些蛋白被认为作为RNA伴侣，刺激U6与U4 snRNA配对，形成U4/U6双链的分子间茎I和茎II，其形成对剪接体功能至关重要。然而，Prp24p和LSm复合物促进U4/U6碱基配对的机制以及Prp24p在原生U6 snRNP中的确切结合位点尚不清楚。在这里，我们研究了纯化的U6 snRNPs中U6 snRNA的二级结构，并将其与裸形式进行了比较。利用RNA结构探测技术，我们证明了在U6 snRNP中，U6 snRNA的大部分内部区域是未配对的，并且不受结合Prp24p的化学修饰。这些U6核苷酸中的一些可用于碱基配对相互作用，因为只有它们的糖主链与Prp24p接触。因此，Prp24p可以将它们呈现给U4 snRNA，并促进U4/U6茎i的形成。我们发现，在天然颗粒中，3'茎环不受U6蛋白的强烈结合。然而，与裸露的U6 snRNA中的3'茎环相比，它具有更开放的构象，这将有利于与U4 snRNA形成茎11。我们的数据表明，Prp24p和LSm复合物的结合赋予了U6核苷酸有利于U4/U6碱基配对的构象。有趣的是，我们发现酵母U6 snRNA在天然snRNPs中的开放结构也可以被人类U6和U6atac snRNAs所采用。(c) 2006 Elsevier Ltd.版权所有。
The U6 small nuclear RNA (snRNA) undergoes major conformational changes during the assembly of the spliceosome and catalysis of splicing. It associates with the specific protein Prp24p, and a set of seven LSm2p-8p proteins, to form the U6 small nuclear ribonucleoprotein (snRNP). These proteins have been proposed to act as RNA chaperones that stimulate pairing of U6 with U4 snRNA to form the intermolecular stem I and stem II of the U4/U6 duplex, whose formation is essential for spliceosomal function. However, the mechanism whereby Prp24p and the LSm complex facilitate U4/U6 base-pairing, as well as the exact binding site(s) of Prp24p in the native U6 snRNP, are not well understood. Here, we have investigated the secondary structure of the U6 snRNA in purified U6 snRNPs and compared it with its naked form. Using RNA structure-probing techniques, we demonstrate that within the U6 snRNP a large internal region of the U6 snRNA is unpaired and protected from chemical modification by bound Prp24p. Several of these U6 nucleotides are available for base-pairing interaction, as only their sugar backbone is contacted by Prp24p. Thus, Prp24p can present them to the U4 snRNA and facilitate formation of U4/U6 stem I. We show that the 3' stem-loop is not bound strongly by U6 proteins in native particles. However, when compared to the 3' stem-loop in the naked U6 snRNA, it has a more open conformation, which would facilitate formation of stem 11 with the U4 snRNA. Our data suggest that the combined association of Prp24p and the LSm complex confers upon U6 nucleotides a conformation favourable for U4/U6 base-pairing. Interestingly, we find that the open structure of the yeast U6 snRNA in native snRNPs can also be adopted by human U6 and U6atac snRNAs. (c) 2006 Elsevier Ltd. All rights reserved.