Conversion of pre-RISC to holo-RISC by Ago2 during assembly of RNAi complexes.
In the Drosophila RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) direct Argonaute2 (Ago2), an endonuclease, within the RNA-induced silencing complex (RISC) to cleave complementary mRNA targets. In vitro studies have shown that, for each siRNA duplex, RISC retains only one strand, the guide, and releases the other, the passenger, to form a holo-RISC complex. Here, we have isolated a new Ago2 mutant allele and provide, for the first time, in vivo evidence that endogenous Ago2 slicer activity is important to mount an RNAi response in Drosophila. We demonstrate in vivo that efficient removal of the passenger strand from RISC requires the cleavage activity of Ago2. We have also identified a new intermediate complex in the RISC assembly pathway, pre-RISC, in which Ago2 is stably bound to double-stranded siRNA.
Kim
Kevin
K
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208, USA.
Lee
Young Sik
YS
Carthew
Richard W
RW
D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D060565:Argonaute Proteins; D004330:Drosophila / Q000201:enzymology* / Q000235:genetics; D029721:Drosophila Proteins / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
C433807:AGO2 protein, Drosophila; D060565:Argonaute Proteins; D029721:Drosophila Proteins; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex