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: Contribution to journalArticle

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
Pages (from-to)345-355
Number of pages11
JournalMethods in Enzymology
Volume449
Issue numberC
DOIs
Publication statusPublished - 2008

Fingerprint

Guide RNA
RNA Interference
RNA
Solanum melongena
Asparagine
Diptera
Messenger RNA
RNA Cleavage
Gene Silencing
Histidine
Germ Cells
Base Pairing
Testis
Genes
Gene Expression
Antibodies
Assays
Proteins
Association reactions

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Chapter 16 How to Define Targets for Small Guide RNAs in RNA Silencing. A Biochemical Approach. / Siomi, Mikiko C.; Nishida, Kazumichi M.; Siomi, Haruhiko.

In: Methods in Enzymology, Vol. 449, No. C, 2008, p. 345-355.

Research output: Contribution to journalArticle

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