Bacterial Expression of Mouse Argonaute 2 for Functional and Mutational Studies

Abstract

RNA interference (RNAi) is a post-transcriptional gene-silencing process that occurs in many eukaryotic organisms upon intracellular exposure to double-stranded RNA. Argonaute 2 (Ago2) protein is the catalytic engine of mammalian RNAi. It contains a PIWI domain that is structurally related to RNases H and possibly shares with them a two-metal-ion catalysis mechanism. Here we describe the expression in E. coli of mouse Ago2 and testing of its enzymatic activity in a RISC assay, i.e., for the ability to cleave a target RNA in a single position specified by a complementary small interfering RNA (siRNA). The results show that the enzyme can load the siRNA and cleave the complementary RNA in absence of other cellular factors, as described for human Ago2. It was also found that mutation of Arg669, a residue previously proposed to be involved in substrate and/or B metal ion binding, doesn’t affect the enzymatic activity, suggesting that this residue doesn’t belong to the active site.

Molecular mechanisms governing microRNA-125a expression in human hepatocellular carcinoma cells

“…The 869mut construct with the mutated translation start codons described in Fig. 2 was obtained by site-directed mutagenesis with the PCR-based overlap extension method 61 as described 62 . All the reporter constructs were sequenced to confirm their identity.…”