| Chapter title |
Peptide-Based Isolation of Argonaute Protein Complexes Using Ago-APP
|
|---|---|
| Chapter number | 9 |
| Book title |
MicroRNA Detection and Target Identification
|
| Published in |
Methods in molecular biology, April 2017
|
| DOI | 10.1007/978-1-4939-6866-4_9 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-6864-0, 978-1-4939-6866-4
|
| Authors |
Judith Hauptmann, Gunter Meister |
| Editors |
Tamas Dalmay |
| Abstract |
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. |
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