Purified Argonaute2 and an siRNA form recombinant human RISC.
Genetic, biochemical and structural studies have implicated Argonaute proteins as the catalytic core of the RNAi effector complex, RISC. Here we show that recombinant, human Argonaute2 can combine with a small interfering RNA (siRNA) to form minimal RISC that accurately cleaves substrate RNAs. Recombinant RISC shows many of the properties of RISC purified from human or Drosophila melanogaster cells but also has surprising features. It shows no stimulation by ATP, suggesting that factors promoting product release are missing from the recombinant enzyme. The active site is made up of a unique Asp-Asp-His (DDH) motif. In the RISC reconstitution system, the siRNA 5' phosphate is important for the stability and the fidelity of the complex but is not essential for the creation of an active enzyme. These studies demonstrate that Argonaute proteins catalyze mRNA cleavage within RISC and provide a source of recombinant enzyme for detailed biochemical studies of the RNAi effector complex.
Rivas
Fabiola V
FV
Cold Spring Harbor Laboratory, Watson School of Biological Sciences, New York 11724, USA.
Tolia
Niraj H
NH
Song
Ji-Joon
JJ
Aragon
Juan P
JP
Liu
Jidong
J
Hannon
Gregory J
GJ
Joshua-Tor
Leemor
L
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D020816:Amino Acid Motifs; D000818:Animals; D060565:Argonaute Proteins; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D018360:Crystallography, X-Ray; D004331:Drosophila melanogaster; D015852:Eukaryotic Initiation Factor-2; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016914:Ribonuclease H / Q000737:chemistry; D040681:Structural Homology, Protein
C466422:AGO2 protein, human; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D010448:Peptide Initiation Factors; D010710:Phosphates; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D016914:Ribonuclease H