Chapter 16. How to define targets for small guide RNAs in RNA silencing: a biochemical approach.
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.
Siomi
Mikiko C
MC
Keio University School of Medicine, Tokyo, Japan; JST, CREST, Saitama, Japan.
Nishida
Kazumichi M
KM
Siomi
Haruhiko
H
D000818:Animals; D060565:Argonaute Proteins; D015152:Blotting, Northern; D015153:Blotting, Western; D004330:Drosophila; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D039642:Eukaryotic Initiation Factors; D047468:Immunoprecipitation; D008297:Male; D034622:RNA Interference / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000235:genetics / Q000378:metabolism
C489654:AGO1 protein, Drosophila; D060565:Argonaute Proteins; D029721:Drosophila Proteins; D039642:Eukaryotic Initiation Factors; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex