Leeder Wolf-Matthias, Kruse Elisabeth, Göringer H Ulrich
Molecular Genetics, Technical University Darmstadt, Darmstadt, Germany.
Bio Protoc. 2021 Mar 5;11(5):e3935. doi: 10.21769/BioProtoc.3935.
Gene expression within the mitochondria of African trypanosomes and other protozoan organisms relies on a nucleotide-specific RNA-editing reaction. In the process exclusively uridine (U)-nucleotides are site-specifically inserted into and deleted from sequence-deficient primary transcripts to convert them into translatable mRNAs. The reaction is catalyzed by a 0.8 MDa multiprotein complex termed the editosome. Here we describe an improved test to quantitatively explore the catalytic activity of the editosome. The assay uses synthetic, fluorophore-derivatized oligoribonucleotides as editing substrates, which enable the automated electrophoretic separation of the reaction products by capillary electrophoresis (CE) coupled to laser-induced fluorescence (LIF) detection systems. The assay is robust, it requires only nanogram amounts of materials and by using multicapillary CE/LIF-instruments it can be executed in a highly parallel layout. Further improvements include the usage of phosphorothioate-modified and thus RNase-resistant substrate RNAs as well as multiplex-type fluorophore labeling strategies to monitor the U-insertion and U-deletion reaction simultaneously. The assay is useful for investigating the mechanism and enzymology of the editosome. However, it can also be executed in high-throughput to screen for RNA editing-specific inhibitors. .
非洲锥虫及其他原生动物线粒体内的基因表达依赖于一种核苷酸特异性的RNA编辑反应。在此过程中，仅尿苷（U）核苷酸会在序列缺失的初级转录本中进行位点特异性的插入和删除，从而将其转化为可翻译的mRNA。该反应由一个称为编辑体的800 kDa多蛋白复合体催化。本文描述了一种经过改进的测试方法，用于定量探究编辑体的催化活性。该检测方法使用合成的、荧光团衍生化的寡核糖核苷酸作为编辑底物，能够通过与激光诱导荧光（LIF）检测系统联用的毛细管电泳（CE）对反应产物进行自动电泳分离。该检测方法稳定性好，仅需纳克量的材料且通过使用多毛细管CE/LIF仪器可以在高度平行的布局中进行。进一步的改进包括使用硫代磷酸酯修饰从而抗核糖核酸酶（RNase）的底物RNA以及多重荧光团标记策略，以同时监测U插入和U删除反应。该检测方法对于研究编辑体的机制和酶学很有用。然而，它也可以高通量进行，以筛选RNA编辑特异性抑制剂。
