植物中AGO介导的靶标切割和翻译抑制的体外分析

In Vitro Analysis of ARGONAUTE-Mediated Target Cleavage and Translational Repression in Plants.

作者信息

Tomari Yukihide, Iwakawa Hiro-Oki

机构信息

Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, Japan.

Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.

出版信息

Methods Mol Biol. 2017;1640:55-71. doi: 10.1007/978-1-4939-7165-7_4.

DOI:10.1007/978-1-4939-7165-7_4

PMID:28608334

Abstract

MicroRNAs (miRNAs) are endogenous small RNAs, which negatively regulate expression of complementary target genes at the post-transcriptional level. In plants, miRNAs are mainly loaded onto ARGONAUTE1 to form RNA-induced silencing complexes (RISCs), which mediate target mRNA cleavage as well as translational repression. The cell-free system derived from tobacco BY-2 protoplasts has become a powerful tool not only for the analysis of RISC assembly mechanism but also for mechanistic dissection of plant RISC functions. Here we describe the detailed protocols for the preparation of BY-2 cell lysate and the procedure to analyze the dual function of plant RISC-target cleavage and translational repression-in vitro.

摘要

微小RNA（miRNAs）是内源性小RNA，在转录后水平负向调控互补靶基因的表达。在植物中，miRNAs主要装载到AGO1（AGO1蛋白）上以形成RNA诱导沉默复合体（RISCs），介导靶mRNA的切割以及翻译抑制。源自烟草BY-2原生质体的无细胞系统不仅已成为分析RISC组装机制的有力工具，而且还用于对植物RISC功能进行机制剖析。在此，我们描述了制备BY-2细胞裂解物的详细方案以及在体外分析植物RISC切割和翻译抑制双重功能的程序。