Identification Of Proteins Interacting With RISC And The Study Of Their Functions

Objective: To investigate the proteins correlated with RISC using yeast two-hybrid system and further identify their functions.Methods: Coding region for human AGO2 C terminal was cloned into the pSos vector as bait plasmid. Screening of proteins interact with bait protein was carried out using CytoTrap yeast two-hybrid system with the cDNA library from human lung.After the verification of specificity of protein-protein interactions, the interactions between bait and targets were confirmed using immunocoprecipitation and western blot. In vivo, the effects of interactive proteins on RISC activities were investigated by expressing the proteins in RNAi system of human glioblastoma cell line U373.Results: 22 putative positive interactors were found by library screening. After verification of specificity of protein-protein interactions, 7 plasmids remained positive interactors. Using immunocoprecipitation for further confirmation, we ultimately find 4 interactive proteins: Homo sapiens proteasome 26S subunit, ATPase, 3; Homo sapiens DNA fragmentation factor, 45kDa, alpha polypeptide (DFFA), isoform 2; Homo sapiens p53-associated cellular protein (PACT); Homo sapiens retinoblastoma binding protein 6 (RBBP6) , isoform 2. Experiments in vivo proved that Homo sapiens proteasome 26S subunit ATPase 3 increases the activity of RISC and human PACT represses the activity of RISC.