在细胞提取物中用小双链RNA装载Argonaute蛋白。

Loading of Argonaute Protein with Small Duplex RNA in Cellular Extracts.

作者信息

Gagnon Keith T

机构信息

Department of Biochemistry and Molecular Biology, School of Medicine, Southern Illinois University, Carbondale, IL, 62901, USA.

Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale, IL, 62901, USA.

出版信息

Methods Mol Biol. 2016;1421:53-67. doi: 10.1007/978-1-4939-3591-8_6.

DOI:10.1007/978-1-4939-3591-8_6

PMID:26965257

Abstract

Argonaute (Ago) proteins are the minimum core proteins required for executing RNA interference (RNAi) mechanisms of gene regulation. For Ago proteins to regulate gene expression through RNAi they must be loaded, or "programmed," with a single strand of small RNA. Natural small RNAs are typically double-stranded duplexes that require additional factors for efficient and specific loading into Ago proteins. Here, a protocol is described for investigating RNAi programming through loading of human Ago2 using radiolabeled small interfering RNA (siRNA) and HeLa cell extracts. This protocol provides an Ago loading assay to study RNAi programming when starting with crude or partially purified cell extracts. The Ago loading assay should prove useful for studying other Ago proteins using a variety of mammalian cell extracts.

摘要

AGO蛋白是执行基因调控的RNA干扰（RNAi）机制所需的最小核心蛋白。AGO蛋白要通过RNAi调节基因表达，必须用单链小RNA进行装载，即“编程”。天然小RNA通常是双链双链体，需要其他因子才能有效且特异性地装载到AGO蛋白中。本文描述了一种使用放射性标记的小干扰RNA（siRNA）和HeLa细胞提取物，通过装载人AGO2来研究RNAi编程的方法。该方法提供了一种AGO装载测定法，用于在起始材料为粗制或部分纯化的细胞提取物时研究RNAi编程。AGO装载测定法对于使用各种哺乳动物细胞提取物研究其他AGO蛋白应是有用的。