Structural insights into RNA processing by the human RISC loading complex英文文献.pdfVIP

a r t i c l e s Structural insights into RNA processing by the human RISC-loading complex 1,2 3,8 3,4,8 2,8 5 Hong-Wei Wang , Cameron Noland , Bunpote Siridechadilok , David W Taylor , Enbo Ma , Karin Felderer3,5, Jennifer A Doudna3,5–7 Eva Nogales1,3,5 Targeted gene silencing by RNA interference (RNAi) requires loading of a short guide RNA (small interfering RNA (siRNA) or . d microRNA (miRNA)) onto an Argonaute protein to form the functional center of an RNA-induced silencing complex (RISC). e v In humans, Argonaute2 (AGO2) assembles with the guide RNA–generating enzyme Dicer and the RNA-binding protein TRBP r e s to form a RISC-loading complex (RLC), which is necessary for efficient transfer of nascent siRNAs and miRNAs from Dicer to e r AGO2. Here, using single-particle EM analysis, we show that human Dicer has an L-shaped structure. The RLC Dicer’s N-terminal s t DE×H/D domain, located in a short ‘base branch’, interacts with TRBP, whereas its C-terminal catalytic domains in the main body h g i are proximal to AGO2. A model generated by docking the available atomic structures of Dicer and Argonaute homologs into the r l l RLC reconstruction suggests a mechanism for siRNA transfer from Dicer to AGO2. A . c In RNAi and related pathways, guide RNAs of ~21 nucleotides (nt) as well-dispersed, elongated particles with a length of ~20 nm n I , 1 a assemble into large ribonucleoprotein complexes termed RISCs . (Supplementary Fig. 1). Using random conical tilt (RCT) method- c