Regulation Of Rna Interference By Dffa And Psmc3

Objective:RNA interference is a type of gene silencing mediated by siRNA or dsRNA. Such RNAs can cooperate with Argonaute and associated factors into RNA-induced silencing complex (RISC) by combining with their target mRNAs. Finally, RISC can implement cleavage and degradation of the targets. To confirm the association of these proteins with human Arogonaute2, we used coimmunoprecipitation to identify the specificity of interactions. Additionally, the results of fluorescence experiment shows that the novel parters is functionally required to mediate miRNA and siRNA-guided mRNA cleavage in vivo.Methods:We confirmed DFFA and PSMC3 can interacted with hAgo2 specifically in vivo or in vitro by coimmunoprecipitation experiment and GST pull-down experiment. We modified a previously developed GFP-based positive readout reporter system to identify the function of DFFA and PSMC3 in guiding miRNA-specific target RNA cleavage; On the other hand, to determine the functional role of DFFA in siRNA-midiated gene silencing mechanism, we first transfected HeLa cell stable expressing GFP with siRNA targeting DFFA, transfected them 24 hours later with siRNA targeting GFP, and quantified the GFP level. Result:1. We confirmed hAgo2 PIWI can be precipitated by Flag fusion proteins-DFFA and PSMC3, it imply that the domain of hAgo2 has special interaction with the novel factors. The mapping result showed only the whole DFFA or PSMC3 fuse protein can pull down by endogenetic Argonaute2.2. We identificated that hAgo2 PIWI domain can be pull down by GST-fused DFFA, it shows DFFA has special interaction with the domain in vitro.3. GFP reporter system showed fluorescence signal depressed by miR-21 or siRNA can be reliefed by knocking down DFFA or PSMC3 in HeLa cell line.Conclusion:We confirm Argonaute 2 can be pulled down by the full length Flag-fused DFFA and PSMC3 through the experiments in vivo and in vitro. Depletion of DFFA or PSMC3 can relived the in inhibition mediated by miR-21 in vivo or si-GFP in vitro, we supposed the novel proteins interacted with Argonaute2 were component of RISC.