Stojković Vanja, Weinberg David E, Fujimori Danica Galonić
Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA, USA.
Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA.
Methods Mol Biol. 2021;2298:105-122. doi: 10.1007/978-1-0716-1374-0_7.
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.
自由基SAM RNA甲基化酶家族由一大类蛋白质组成，其中只有少数成员具有功能特征。该家族中的几种酶与翻译调控和抗生素敏感性有关，这凸显了它们在细菌生理学中的重要性以及与人类健康的相关性。虽然已显示少数具有特征的酶可修饰多种RNA底物，这表明该家族内潜在的底物范围广泛，但该类中的许多酶尚无已知底物。对于未表征的家族成员，精确了解其RNA底物和修饰位点对于阐明其生物学功能至关重要。在此，我们描述了一种底物鉴定策略，该策略利用了酶与其RNA底物之间基于机制的交联，我们将其命名为单核苷酸分辨率交联和免疫沉淀结合测序突变分析（miCLIP-MaPseq）。使用热稳定的II组内含子逆转录酶（TGIRT）来确定修饰位点的位置，该酶会在交联位点引入错配。
