Mapping of Ago2-GW182 functional interactions.

MicroRNA (miRNA)-mediated posttranscriptional regulation of gene expression has become a major focus in understanding fine-tuning controls in many biological processes. Argonaute 2 protein (Ago2), a core component of RNA-induced silencing complex, directly binds miRNA and functions in both RNAi and miRNA pathways. GW182 is a marker protein of GW bodies (GWB, also known as mammalian P-bodies) and is known to bind the Ago2 protein. This Ago2-GW182 interaction is crucial for Ago2-miRNA-mediated translational silencing as well as the recruitment of Ago2 into GWB. Translational silencing of tethered Ago2 to a 3'UTR reporter requires GW182 for function, whereas tethered GW182 exerts a stronger repression than tethered Ago2 and does not apparently require Ago2. This chapter describes in detail the methods used in mapping Ago2-GW182 interactions.