Martinez Nicole M, Gilbert Wendy V
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
Methods Mol Biol. 2021;2298:379-397. doi: 10.1007/978-1-0716-1374-0_22.
Pseudouridine profiling has revealed many previously unknown sites of the RNA modification pseudouridine (Ψ) in cellular RNAs. All organisms express multiple pseudouridine synthases (PUS) whose RNA targets and mechanisms of targeting remain to be elucidated. Here, we describe a high-throughput in vitro pseudouridylation assay to interrogate pseudouridine status upon incubation with recombinant pseudouridine synthases (PUS) at thousands of RNA sequences of interest in parallel. This approach allows validation of sites provisionally identified in cells, identification of the direct targets of individual PUS, and interrogation of the determinants of target recognition including primary sequence and RNA secondary structure.
假尿苷谱分析揭示了细胞RNA中许多以前未知的RNA修饰假尿苷（Ψ）位点。所有生物体都表达多种假尿苷合酶（PUS），其RNA靶点和靶向机制仍有待阐明。在这里，我们描述了一种高通量体外假尿苷化分析方法，用于在与重组假尿苷合酶（PUS）一起孵育时，同时对数千个感兴趣的RNA序列的假尿苷状态进行检测。这种方法可以验证在细胞中初步鉴定的位点，确定单个PUS的直接靶点，并探究包括一级序列和RNA二级结构在内的靶点识别决定因素。
