Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, AB, Canada.
Methods Mol Biol. 2021;2298:357-378. doi: 10.1007/978-1-0716-1374-0_21.
Posttranscriptional modifications of RNA play an important role in promoting the maturation and functional diversity of many RNA species. Accordingly, understanding the enzymes and mechanisms that underlie RNA modifications is an important aspect in advancing our knowledge of the continually expanding RNA modification field. However, of the more than 160 currently identified RNA modifications, a large portion remains without quantitative detection assays for their biochemical characterization. Here, we describe the tritium release assay as a convenient tool allowing for the quantitative assessment of in vitro RNA pseudouridylation by stand-alone or box H/ACA RNA-guided pseudouridine synthases. This assay enables quantification of RNA pseudouridylation over a time course to effectively compare pseudouridylation activity between different substrates and/or different recombinant enzymes as well as to determine kinetic parameters. With the help of a quench-flow apparatus, the tritium release assay can be adapted for rapid kinetic measurements under single-turnover conditions to dissect reaction mechanisms. As examples, we show the formation of pseudouridines by a reconstituted Saccharomyces cerevisiae H/ACA small ribonucleoprotein (snoRNP) and an Escherichia coli stand-alone pseudouridine synthase.
RNA 的转录后修饰在促进许多 RNA 物种的成熟和功能多样性方面发挥着重要作用。因此，了解 RNA 修饰所依赖的酶和机制是深入了解不断扩展的 RNA 修饰领域的重要方面。然而，在目前已鉴定的 160 多种 RNA 修饰中，其中很大一部分仍然缺乏用于其生化特征描述的定量检测方法。在这里，我们描述了氚释放测定法作为一种方便的工具，可用于通过独立或盒 H/ACA RNA 指导的假尿嘧啶合酶体外定量评估 RNA 假尿嘧啶化。该测定法可在时间过程中定量 RNA 假尿嘧啶化，从而有效地比较不同底物和/或不同重组酶之间的假尿嘧啶化活性，并确定动力学参数。借助快速混合装置，氚释放测定法可以适应单轮条件下的快速动力学测量，以剖析反应机制。作为示例，我们展示了由重组酿酒酵母 H/ACA 小核仁核糖核蛋白 (snoRNP) 和大肠杆菌独立假尿嘧啶合酶形成的假尿嘧啶。
