Peptide-Based Isolation of Argonaute Protein Complexes Using Ago-APP
Argonaute (Ago) proteins bind small RNAs such as microRNAs (miRNAs) or short interfering RNAs (siRNAs), which guide them to distinct mRNAs for post-transcriptional gene silencing. Mammalian miRNA-guided gene silencing pathways mainly lead to translational repression and mRNA destabilization. To facilitate these processes, Ago proteins bind members of the GW protein family, which form central interaction platforms for the recruitment of downstream effector proteins. GW proteins use tryptophane residues (W) to bind to the surface of Ago proteins. This high affinity interaction is retained when a short, GST-fused GW peptide is used in biochemical pull-down experiments—an approach referred to as “Ago Affinity Purification by Peptides” (Ago-APP). Since the binding interface is conserved among different paralogues and different species, Ago-APP represents a universal tool to purify Ago proteins and associated small RNAs using samples from species with conserved miRNA pathways.
Key wordsArgonaute proteins Small RNAs miRNAs Ago-APP
Our research is supported by grants from the Deutsche Forschungsgemeinschaft (SFB 960, FOR2127), the European Research Council (ERC grant 242792 “sRNAs”, ITN RNATrain), the Bavarian Genome Research Network (BayGene), the German Cancer Aid and the Bavarian Systems-Biology Network (BioSysNet).