A Role For DFFA And PSMC3Interactes With Human Argonaute2in MicroRNA Function

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. In previous reporter, we used human argonaute2structural domain of PIWI as the bait of CytoTrap(?) yeast two-hybrid system with the cDNA library from human lung screening a two novel proteins DFFA and PSMC3. To confirm the association of these proteins with human Arogonaute2, we used coimmunoprecipitation to identify the specificity of interactions. And it is important to investigate the proteins interacting with Ago2to learn the RNAi mechanism.Methods:We confirmed DFFA and PSMC3can interacted with hAgo2specifically in vivo by coimmunoprecipitation experiment. We modified a previously developed EGFP-RAS positive reporter system to identify the function of DFFA and PSMC3in repressing miRNA-mediated translation.Resμlt:1. We confirmed hAgo2PIWI can be precipitated by Flag fusion proteins-DFFA and PSMC3, it imply that the domain of hAgo2has special interaction with the novel factors. The mapping resμlt showed only the whole DFFA or PSMC3fuse protein can pμll down by endogenetic Argonaute2.2. GFP reporter system showed fluorescence signal depressed by let-7can be reliefed by knocking down DFFA or PSMC3in HeLa cell line.Conclusion:We confirm Argonaute2can be pμlled down by the fμll length Flag-fused DFFA and PSMC3throμthe experiments in vivo. Depletion of DFFA or PSMC3can reduced the in inhibition mediated by let-7in vivo, we supposed the two novel proteins interacted with Argonaute2were the components of RISC.