RNA Remodeling by RNA Chaperones Monitored by RNA Structure Probing.
RNA Remodeling by RNA Chaperones Monitored by RNA Structure Probing.
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通过 RNA 结构探测监测 RNA 伴侣的 RNA 重塑
DOI：
10.1007/978-1-0716-0231-7_11
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发表时间：
2020
影响因子：
--
通讯作者：
Behrens SE
中科院分区：
文献类型：
--
作者：
Friedrich S;Schmidt T;Behrens SE
RNA结构探测能够通过已建立的程序（例如基于酶或化学的单链或双链区域检测）表征RNA二级结构。一种特定类型的应用涉及检测由具有RNA伴侣活性的蛋白质诱导的RNA结构和构象的变化。本章概述了一个协议来分析RNA结构在体外的RNA结合蛋白与RNA伴侣活性的存在下。为此，我们使用甲基化剂硫酸二甲酯（DMS）和1-环己基-3-（2-吗啉代乙基）碳二亚胺甲基对甲苯磺酸酯（CMCT）。DMS和CMCT特异性修饰不参与碱基配对或三级结构氢键并且不受配体（例如蛋白质）保护的核苷酸。通过引物延伸鉴定修饰的碱基。作为一个例子，我们描述了RNA结合蛋白AUF 1亚型的RNA伴侣活性如何诱导病毒基因组环化和病毒复制所需的黄病毒RNA开关，本章包括RNA体外合成、DNA引物的32 P-5′-末端标记、引物延伸以及分析凝胶的制备和运行的综合方案。所描述的方法应该适用于任何其他感兴趣的RNA和蛋白质，以鉴定蛋白质指导的RNA重塑。
RNA structure probing enables the characterization of RNA secondary structures by established procedures such as the enzyme- or chemical-based detection of single- or double-stranded regions. A specific type of application involves the detection of changes of RNA structures and conformations that are induced by proteins with RNA chaperone activity. This chapter outlines a protocol to analyze RNA structures in vitro in the presence of an RNA-binding protein with RNA chaperone activity. For this purpose, we make use of the methylating agents dimethyl sulfate (DMS) and 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMCT). DMS and CMCT specifically modify nucleotides that are not involved in base-pairing or tertiary structure hydrogen bonding and that are not protected by a ligand such as a protein. Modified bases are identified by primer extension. As an example, we describe how the RNA chaperone activity of an isoform of the RNA-binding protein AUF1 induces the flaviviral RNA switch required for viral genome cyclization and viral replication.This chapter includes comprehensive protocols for in vitro synthesis of RNA,32P-5′-end labeling of DNA primers, primer extension, as well as the preparation and running of analytical gels. The described methodology should be applicable to any other RNA and protein of interest to identify protein-directed RNA remodeling.
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影响因子：
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作者：
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通讯作者：
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通讯作者：
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