miCLIP-MaPseq Identifies Substrates of Radical SAM RNA-Methylating Enzyme Using Mechanistic Cross-Linking and Mismatch Profiling.
miCLIP-MaPseq Identifies Substrates of Radical SAM RNA-Methylating Enzyme Using Mechanistic Cross-Linking and Mismatch Profiling.
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DOI：
10.1007/978-1-0716-1374-0_7
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Fujimori DG
中科院分区：
文献类型：
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作者：
Stojković V;Weinberg DE;Fujimori DG
自由基SAM RNA甲基化酶家族包括一大组蛋白质，其仅包含少数功能特征成员。该家族中的几种酶与翻译和抗生素敏感性的调节有关，强调了它们在细菌生理学中的重要性及其与人类健康的相关性。虽然很少有表征的酶已被证明可以修饰不同的RNA底物，突出了家族内潜在的广泛底物范围，但这类酶中的许多酶没有已知的底物。精确了解RNA底物和修饰位点的未表征的家庭成员是很重要的解开他们的生物学功能。在这里，我们描述了一种底物识别策略，该策略利用了酶与其RNA底物之间基于机制的交联，我们将其命名为单个核苷酸分辨率交联和免疫沉淀结合突变分析与测序（miCLIP-MaPseq）。使用热稳定性II组内含子逆转录酶（TGIRT）实现修饰位点的位置的鉴定，所述热稳定性II组内含子逆转录酶在交联位点处引入错配。
The family of radical SAM RNA-methylating enzymes comprises a large group of proteins that contains only a few functionally characterized members. Several enzymes in this family have been implicated in the regulation of translation and antibiotic susceptibility, emphasizing their significance in bacterial physiology and their relevance to human health. While few characterized enzymes have been shown to modify diverse RNA substrates, highlighting potentially broad substrate scope within the family, many enzymes in this class have no known substrates. The precise knowledge of RNA substrates and modification sites for uncharacterized family members is important for unraveling their biological function. Here, we describe a strategy for substrate identification that takes advantage of mechanism-based cross-linking between the enzyme and its RNA substrates, which we named individual-nucleotide-resolution cross-linking and immunoprecipitation combined with mutational profiling with sequencing (miCLIP-MaPseq). Identification of the position of the modification site is achieved using thermostable group II intron reverse transcriptase (TGIRT), which introduces a mismatch at the site of the cross-link.