Biochemical isolation of Argonaute protein complexes by Ago-APP

Significance Small RNA-guided gene-silencing pathways regulate fundamental cellular processes. Small RNAs such as microRNAs (miRNAs) directly bind to a member of the Argonaute (Ago) protein family. In animals, Ago proteins interact with a member of the GW protein family (referred to as TNRC6A-C). Based on an Ago-interacting TNRC6 peptide, we have developed a method allowing for the efficient isolation and characterization of Ago protein complexes from any animal organism. We refer to this method as “Ago protein Affinity Purification by Peptides.” Our approach also allows for the identification of Ago-bound small RNAs as well as mRNAs. Expression of this peptide in living cells leads to global miRNA inactivation, thus providing a powerful tool to study miRNA function on various levels. During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as “Ago protein Affinity Purification by Peptides“ (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells.

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