Abstract
MicroRNAs (miRNAs) are a large class of small noncoding RNAs that regulate the expression of distinct target mRNAs. miRNAs are incorporated into Argonaute (AGO) proteins and guide them to their target mRNAs. Subsequently, AGO proteins recruit a member of the glycine-tryptophan-rich (GW) protein family by direct protein-protein interaction. GW proteins coordinate all downstream processes leading to robust and efficient gene silencing. A short peptide of GW proteins comprising the AGO interaction motif can be used to biochemically isolate endogenous AGO protein complexes. Furthermore, within a cell such a peptide competes with endogenous GW proteins for AGO binding and thus can be used as potent inhibitor of the miRNA pathway. Here, we describe a method that utilizes a GW-based polypeptide (T6B-assay) to validate miRNA-mRNA interactions in tissue culture systems.
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