title: Reduced levels of Ago2 expression result in increased siRNA competition in mammalian cells.
abstract: Administration of small interfering RNAs (siRNAs) leads to degradation of specific mRNAs utilizing the cellular RNA interference (RNAi) machinery. It has been demonstrated that co-administration of siRNAs may lead to attenuation of activity of one of the siRNAs. Utilizing antisense and siRNA-mediated RNA-induced silencing complex (RISC) gene reduction we show that siRNA competition is correlated with differences in the cellular expression levels of Ago2, while levels of other RISC proteins have no effect on competition. We also show that under certain conditions siRNA competition rather than reduction of cellular RISC levels may be responsible for apparent reduction in siRNA activity. Furthermore, exploiting siRNA competition, we show that the RISC pathway loads and results in detectable cleavage of the target RNA in approximately 2 h after transfection. The RISC pathway is also capable of being reloaded even in the absence of new protein synthesis. RISC reloading and subsequent induction of detectable cleavage of a new target RNA, requires about 9-12 h following the initial transfection.
authors: Isis Pharmaceuticals. 1896 Rutherford Road, Carlsbad, CA 92008, USA. tvickers@isisph.com
authors: Timothy A
authors: TA
authors: Vickers
authors: Walt F
authors: WF
authors: Lima
authors: Josh G
authors: JG
authors: Nichols
authors: Stanley T
authors: ST
authors: Crooke
mesh_terms: D060565:Argonaute Proteins; D002460:Cell Line; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000378:metabolism*; D043244:Ribonuclease III / Q000378:metabolism
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D043244:Ribonuclease III