Chapter 16 How to Define Targets for Small Guide RNAs in RNA Silencing. A Biochemical Approach

Mikiko C. Siomi, Kazumichi M. Nishida, Haruhiko Siomi

Research output: Chapter in Book/Report/Conference proceedingChapter

3 Citations (Scopus)

Abstract

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.

Original languageEnglish
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages345-355
Number of pages11
EditionC
DOIs
Publication statusPublished - 2008 Jan 1

Publication series

NameMethods in Enzymology
NumberC
Volume449
ISSN (Print)0076-6879

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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