Loading of Argonaute protein with small duplex RNA in cellular extracts

Abstract

Argonaute (Ago) proteins are the minimum core proteins required for executing RNA interference (RNAi) mechanisms of gene regulation. For Ago proteins to regulate gene expression through RNAi they must be loaded, or “programmed,” with a single strand of small RNA. Natural small RNAs are typically doublestranded duplexes that require additional factors for efficient and specific loading into Ago proteins. Here, a protocol is described for investigating RNAi programming through loading of human Ago2 using radiolabeled small interfering RNA (siRNA) and HeLa cell extracts. This protocol provides an Ago loading assay to study RNAi programming when starting with crude or partially purified cell extracts. The Ago loading assay should prove useful for studying other Ago proteins using a variety of mammalian cell extracts.