Salt-Dependent Modulation of the RNA Chaperone Activity of RNA-Binding Protein La.
Salt-Dependent Modulation of the RNA Chaperone Activity of RNA-Binding Protein La.
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RNA 结合蛋白 La 的 RNA 伴侣活性的盐依赖性调节。
DOI：
10.1007/978-1-0716-0231-7_7
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Heise,Tilman
中科院分区：
文献类型：
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作者：
Sommer,Gunhild;Sendlmeier,Christina;Heise,Tilman
众所周知，RNA 结合蛋白 La 具有 RNA 伴侣活性。最近的研究表明，La 蛋白具有两个不同的 RNA 伴侣结构域（RCD-A 和 RCD-B），有助于不同组 RNA 分子的结构变化，例如 RNA 聚合酶 III 转录本（例如前 tRNA、U6 snRNA）、细胞信使和病毒 RNA。在本协议中，我们重点关注位于 La 羧基末端结构域的 RNA 伴侣结构域 RCD-B。研究表明，该 RNA 伴侣结构域有助于折叠在细胞周期蛋白 D1 和 Bcl2 mRNA 5'-非翻译区域中的预测 RNA 发夹的结构变化。除了以 ATP 依赖性方式参与解链 RNA 发夹结构的 RNA 解旋酶之外，RNA 伴侣以 ATP 无关的方式实现类似的功能。为了研究La的RNA伴侣活性，我们建立了基于La依赖分子信标的RNA伴侣测定法，并系统地测试了各种盐条件。在此，我们描述了研究 RNA 伴侣的盐依赖性的测定格式和设计。该方案可以轻松适应测试其他 RNA 结合蛋白的 RNA 伴侣活性并优化测定条件。
It is well established that the RNA-binding protein La has RNA chaperone activity. Recent work suggests that the La protein has two distinct RNA chaperone domains (RCD-A and RCD-B) assisting structural changes in diverse groups of RNA molecules such as RNA polymerase III transcripts (e.g., pre-tRNA, U6 snRNA), cellular messenger, and viral RNAs. In this protocol we focus on the RNA chaperone domain RCD-B, which is located in the carboxy-terminal domain of La. It has been shown that this RNA chaperone domain assists structural changes in predicted RNA hairpins folded in the 5′-untranslated regions of cyclin D1 and Bcl2 mRNAs. Besides RNA helicases, which are implicated in melting RNA hairpin structures in an ATP-dependent manner, RNA chaperones fulfil a similar function in an ATP-independent manner. Aiming to study the RNA chaperon activity of La, we established a La-dependent molecular beacon-based RNA chaperone assay and systematically tested the various salt conditions. Herein we describe the assay format and design to study the salt dependency of RNA chaperones. This protocol can be easily adapted to test the RNA chaperone activity of other RNA-binding proteins and to optimize assay conditions.