Purified Argonaute2 and an siRNA form recombinant human RISC英文文献.pdfVIP

A R T I C L E S Purified Argonaute2 and an siRNA form recombinant b m s human RISC n / m o c . 1,3 1–3 1–3 1 1 1 e Fabiola V Rivas , Niraj H Tolia , Ji-Joon Song , Juan P Aragon , Jidong Liu , Gregory J Hannon r u 1,2 t Leemor Joshua-Tor a n . w w Genetic, biochemical and structural studies have implicated Argonaute proteins as the catalytic core of the RNAi effector w / / : complex, RISC. Here we show that recombinant, human Argonaute2 can combine with a small interfering RNA (siRNA) to p t t form minimal RISC that accurately cleaves substrate RNAs. Recombinant RISC shows many of the properties of RISC purified h from human or Drosophila melanogaster cells but also has surprising features. It shows no stimulation by ATP, suggesting that p u factors promoting product release are missing from the recombinant enzyme. The active site is made up of a unique Asp-Asp- o r His (DDH) motif. In the RISC reconstitution system, the siRNA 5′ phosphate is important for the stability and the fidelity of the G g complex but is not essential for the creation of an active enzyme. These studies demonstrate that Argonaute proteins catalyze n i mRNA cleavage within RISC and provide a source of recombinant enzyme for detailed biochemical studies of the RNAi h s i effector complex. l b u P e r RNA interference or RNAi denotes a group of sequence-specific gene like region. Furthermore, the P. furiosus protein contains conserved u t a silencing pathways that can inhibit gene expression through a variety aspartates, which in related nucleases and transposases are important N 5 of different effector mechanisms1. RNAi is initiated upon the produc- for catalytic activity. The crystal structure of a Pi