Liu Xuhang, Mourelatos Zissimos
Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104-6100, USA.
Methods Mol Biol. 2015;1206:39-51. doi: 10.1007/978-1-4939-1369-5_4.
MicroRNAs (miRNAs) are an important class of small RNAs that regulate gene expression posttranscriptionally through the microRNP (miRNP)/RNA-induced silencing complex (RISC). The core component of miRNPs is an Argonuate protein that directly binds to a miRNA. In mammals, most miRNPs are assembled through the miRNA loading complex (miRLC), which is composed of Dicer, Ago, and TRBP. miRLC processes miRNA precursors (pre-miRNAs) into miRNA duplexes and loads miRNA duplexes to Ago. Here, we describe a native gel analysis system for detecting miRNPs assembled with pre-miRNAs from mammalian lysates that ectopically express Ago2. The methods presented here provide a powerful tool for further dissecting miRNP assembly pathways in mammals.
微小RNA（miRNA）是一类重要的小RNA，它们通过微小RNA核糖核蛋白（miRNP）/RNA诱导沉默复合体（RISC）在转录后水平调节基因表达。miRNP的核心成分是一种直接与miRNA结合的AGO蛋白。在哺乳动物中，大多数miRNP是通过由Dicer、AGO和TRBP组成的miRNA装载复合体（miRLC）组装而成的。miRLC将miRNA前体（pre-miRNA）加工成miRNA双链体，并将miRNA双链体装载到AGO上。在这里，我们描述了一种天然凝胶分析系统，用于检测从异位表达AGO2的哺乳动物裂解物中与pre-miRNA组装而成的miRNP。本文介绍的方法为进一步剖析哺乳动物中miRNP组装途径提供了一个强大的工具。
