Association of Argonaute proteins and microRNAs can occur after cell lysis

Abstract

MicroRNA (miRNA) target identification is a challenging but important endeavor. Global analyses of the direct mRNA targets of miRNAs have relied heavily upon immunopurification techniques, wherein a core protein component of the miRNA–protein complex, Argonaute (Ago), is immunoprecipitated to isolate associated RNAs. This approach involves the assumption that the selected RNAs were bound to the Ago protein in vivo and that the methodology did not significantly perturb endogenous interactions or produce novel interaction artifacts. To test whether RNAs that coimmunoprecipitate with human Ago were bound in vivo or could associate post-cell lysis, we used an experimental approach that distinguishes between these two origins of interaction. We show that a transfected miRNA mimic, but not a plasmid-expressed miRNA, can interact with human Ago proteins post-lysis. Our results have important implications for the design of miRNP immunoprecipitation experiments.