Induction and biochemical purification of RNA-induced silencing complex from Drosophila S2 cells.
The discovery of RNA interference (RNAi) has greatly simplified the process of suppressing genes in many experimental systems, including Caenorhabditis elegans, Drosophila, and mammalian cells. A sequence-specific nuclease complex, called the RNA-induced silencing complex (RISC), can be purified from cells undergoing RNAi. RISC shows RNase activity when exposed to RNAs homologous to the input double-stranded RNA (dsRNAs) but lacks activity in the presence of nontargeted RNAs. We describe the induction of RNAi by dsRNA in cultured Drosophila Schneider-2 (S2) cells and detail procedures for RISC purification from these cells. This purification approach has allowed us to identify several RISC components, including siRNAs, Argo naute 2 (Ago-2), Drosophila Fragile X related protein (dFXR), Vasa intronic gene (VIG), and the micrococcal nuclease family member Tudor-SN (Drosophila CG7008). RNAi is carried out by an endogenous pathway important for normal development in many organisms. In fact, organisms express hundreds of different microRNAs (miRNAs), small hairpin RNAs that function through the RNAi pathway to suppress expression of endogenous genes. The function of miRNAs is poorly understood, and most of their targets are unknown. Purified RISC complexes contain short interfering RNAs and endogenously expressed miRNAs and will be useful for studying many aspects of the RNAi machinery.
Caudy
Amy A
AA
Cold Spring Harbor Laboratory, Watson School of Biological Sciences, Cold Spring Harbor, NY, USA.
Hannon
Gregory J
GJ
D000818:Animals; D002460:Cell Line; D002852:Chromatography, Ion Exchange / Q000379:methods; D004331:Drosophila melanogaster; D008967:Molecular Biology / Q000379:methods; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics* / Q000302:isolation & purification*
D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering