Argonaute2, a link between genetic and biochemical analyses of RNAi.
Double-stranded RNA induces potent and specific gene silencing through a process referred to as RNA interference (RNAi) or posttranscriptional gene silencing (PTGS). RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific, multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs ( approximately 22 nucleotides) derived from the double-stranded RNA trigger, but the protein components of this activity are unknown. Here, we report the biochemical purification of the RNAi effector nuclease from cultured Drosophila cells. The active fraction contains a ribonucleoprotein complex of approximately 500 kilodaltons. Protein microsequencing reveals that one constituent of this complex is a member of the Argonaute family of proteins, which are essential for gene silencing in Caenorhabditis elegans, Neurospora, and Arabidopsis. This observation begins the process of forging links between genetic analysis of RNAi from diverse organisms and the biochemical model of RNAi that is emerging from Drosophila in vitro systems.
Hammond
S M
SM
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
Boettcher
S
S
Caudy
A A
AA
Kobayashi
R
R
Hannon
G J
GJ
D000595:Amino Acid Sequence; D000818:Animals; D060565:Argonaute Proteins; D002460:Cell Line; D004330:Drosophila; D029721:Drosophila Proteins; D004722:Endoribonucleases / Q000378:metabolism; D020868:Gene Silencing; D017344:Genes, Insect; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex; D012091:Repetitive Sequences, Nucleic Acid; D043244:Ribonuclease III; D014162:Transfection
C433807:AGO2 protein, Drosophila; D060565:Argonaute Proteins; D029721:Drosophila Proteins; D019476:Insect Proteins; D012330:RNA, Double-Stranded; D034743:RNA-Induced Silencing Complex; D004722:Endoribonucleases; D043244:Ribonuclease III