Siomi Mikiko C, Nishida Kazumichi M, Siomi Haruhiko
Keio University School of Medicine, Tokyo, Japan; JST, CREST, Saitama, Japan.
Methods Enzymol. 2008;449:345-55. doi: 10.1016/S0076-6879(08)02416-6.
RNA silencing involves various forms of sequence-specific gene silencing triggered by small RNAs. In RNA silencing, Argonautes are crucial protein components that are directed to the target messenger RNAs (mRNAs) through their association with small RNAs by base pairing. Argonautes repress the expression of the target genes at posttranscriptional levels. Full complementarity between a small RNA and its target mRNA results in Argonaute-mediated cleavage ("slicing") of the target mRNA. The D-D-H (asparagine-asparagine-histidine) triad that exists in the PIWI domain of Argonautes is the catalytic center for rendering their target cleavage ("slicer") activity. This chapter describes in vitro target RNA cleavage assays using Aubergine in a complex form with PIWI-interacting RNAs. Aubergine is one of the Argonautes expressed primarily in fly germ lines and is immunopurified from fly testes using the specific antibody against it. The method discussed is useful for defining targets for the small RNAs that function in RNA silencing.
RNA沉默涉及由小RNA触发的各种形式的序列特异性基因沉默。在RNA沉默中，AGO蛋白是关键的蛋白质组分，它们通过与小RNA碱基配对而与靶信使RNA（mRNA）结合。AGO蛋白在转录后水平抑制靶基因的表达。小RNA与其靶mRNA之间的完全互补导致AGO蛋白介导的靶mRNA切割（“切割”）。AGO蛋白PIWI结构域中存在的D-D-H（天冬酰胺-天冬酰胺-组氨酸）三联体是赋予其靶切割（“切割酶”）活性的催化中心。本章描述了使用与PIWI相互作用RNA形成复合物形式的茄子进行体外靶RNA切割试验。茄子是主要在果蝇种系中表达的AGO蛋白之一，并使用针对它的特异性抗体从果蝇睾丸中免疫纯化。所讨论的方法对于确定在RNA沉默中起作用的小RNA的靶标很有用。
