PSMC3and DFFA Regulate The RNAi Through Interaction With Human Argonaute2

Objective:RNA interference (RNAi) has emerged as a new means for controlling gene expression at the transcriptional and post-transcriptional levels. Exogenous dsRNA, siRNAs and endogenous miRNAs play a role of trigger in the course of RNAi. RNA-induced silencing complex (RISC) is the effector of RNAi. AG02is the essential protein component of RISC. The Ago-guide RNA complex binds and cleaves or represses the translation of the target RNA. So it is important to investigate the proteins interacting with Ago2to learn the RNAi mechanism.Methods:1. Immunofluorescence is used to observe the colacation of DFFA-myc or PSMC3-myc and AG02in the A549cells.2. Cotransfection of pcDNA3/GFP,GFP-siRNA and pcDNA3myc/DFFA or DFFA-siRNA or pcDNA3myc/PSMC3or PSMC3-siRNA was used to detect theeffect on RNAi when overexpress or defect of DFFA or PSMC3.3. In vitro cleavage of Target mRNA using cell lysis was carried out to detect the effct of DFFA or PSMC3on RNAi.Results:1. DFFA or PSMC3and AG02colacates in cytoplasm.2. Overexpress of DFFA or PSMC3can not enhance the effect of RNAi while defect of them can inhibit the effect of RNAi obviously.3. Overexpress of DFFA or PSMC3can not enhance the cleavage of target mRNA while defect of them can inhibit the effect obviously.Conclusions:DFFA or PSMC3interacts with AG02in cells. DFFA seems to function as a chaperone for AG02. PSMC3maybe acts as the ATPase needed in the course of RNAi or the proteasome which maybe acts through a post-initiation repression mechanism.