RISC is a 5' phosphomonoester-producing RNA endonuclease.
Gene silencing in the process of RNA interference is mediated by a ribonucleoprotein complex referred to as RNA-induced silencing complex (RISC). Here we describe the molecular mechanism of target RNA cleavage using affinity-purified minimal RISC from human cells. Cleavage proceeds via hydrolysis and the release of a 3'-hydroxyl and a 5'-phosphate terminus. Substitution of the 2'-hydroxyl group at the cleavage site by 2'-deoxy had no significant effect, suggesting that product release and/or a conformational transition rather than a chemical step is rate-limiting. Substitution by 2'-O-methyl at the cleavage site substantially reduced cleavage, which is presumably due to steric interference. Mutational analysis of the target RNA revealed that mismatches across from the 5' or the 3' end of the siRNA had little effect and that substrate RNAs as short as 15 nucleotides were cleaved by RISC.
Martinez
Javier
J
Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY 10021, USA.
Tuschl
Thomas
T
D001665:Binding Sites; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D009154:Mutation; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000378:metabolism*; D013379:Substrate Specificity
D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D012313:RNA