Trotman Jackson B, Schoenberg Daniel R
Center for RNA Biology, The Ohio State University, Columbus, Ohio, USA.
Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, Ohio, USA.
Bio Protoc. 2018 Mar 20;8(6). doi: 10.21769/BioProtoc.2767.
Methyltransferases that methylate the guanine-N7 position of the mRNA 5' cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing samples with a [P]G-capped RNA substrate and S-adenosylmethionine (SAM) to produce RNAs with N7-methylated caps. The extent of cap methylation is then determined by P1 nuclease digestion, thin-layer chromatography (TLC), and phosphorimaging. The protocol described here includes additional steps for generating the [P]G-capped RNA substrate and for preparing nuclear and cytoplasmic extracts from mammalian cells. This assay is also applicable to analyzing the cap methyltransferase activity of other biological samples, including recombinant protein preparations and fractions from analytical separations and immunoprecipitation/pulldown experiments.
使mRNA 5'帽结构的鸟嘌呤N7位甲基化的甲基转移酶在真核生物中普遍存在，并且通常由病毒编码。在这里，我们提供了一个详细的方案，用于对生物样品的RNA帽甲基转移酶活性进行生化分析。该测定法包括将含帽甲基转移酶的样品与[P]G帽化的RNA底物和S-腺苷甲硫氨酸（SAM）一起孵育，以产生具有N7甲基化帽的RNA。然后通过P1核酸酶消化、薄层色谱（TLC）和磷成像来确定帽甲基化的程度。这里描述的方案包括生成[P]G帽化的RNA底物以及从哺乳动物细胞制备核提取物和细胞质提取物的额外步骤。该测定法也适用于分析其他生物样品的帽甲基转移酶活性，包括重组蛋白质制剂以及来自分析分离和免疫沉淀/下拉实验的组分。
