Advertisement

Peptide-Based Isolation of Argonaute Protein Complexes Using Ago-APP

  • Judith Hauptmann
  • Gunter MeisterEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1580)

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.

Key words

Argonaute proteins Small RNAs miRNAs Ago-APP 

Notes

Acknowledgments

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).

References

  1. 1.
    Meister G, Tuschl T (2004) Mechanisms of gene silencing by double-stranded RNA. Nature 431:343–349CrossRefPubMedGoogle Scholar
  2. 2.
    Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136:215–233CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Jinek M, Doudna JA (2009) A three-dimensional view of the molecular machinery of RNA interference. Nature 457:405–412CrossRefPubMedGoogle Scholar
  4. 4.
    Ipsaro JJ, Joshua-Tor L (2015) From guide to target: molecular insights into eukaryotic RNA-interference machinery. Nat Struct Mol Biol 22:20–28CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Meister G et al (2004) Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol Cell 15:185–197CrossRefPubMedGoogle Scholar
  6. 6.
    Liu J et al (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305:1437–1441CrossRefPubMedGoogle Scholar
  7. 7.
    Nakanishi K et al (2013) Eukaryote-specific insertion elements control human ARGONAUTE slicer activity. Cell Rep 3:1893–1900CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Hauptmann J et al (2013) Turning catalytically inactive human Argonaute proteins into active slicer enzymes. Nat Struct Mol Biol 20:814–817CrossRefPubMedGoogle Scholar
  9. 9.
    Hauptmann J et al (2014) Generation of catalytic human Ago4 identifies structural elements important for RNA cleavage. RNA 20:1532–1538CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Faehnle CR et al (2013) The making of a slicer: activation of human Argonaute-1. Cell Rep 3:1901–1909CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Schurmann N et al (2013) Molecular dissection of human Argonaute proteins by DNA shuffling. Nat Struct Mol Biol 20:818–826CrossRefPubMedGoogle Scholar
  12. 12.
    Jonas S, Izaurralde E (2015) Towards a molecular understanding of microRNA-mediated gene silencing. Nat Rev Genet 16:421–433CrossRefPubMedGoogle Scholar
  13. 13.
    Pfaff J, Meister G (2013) Argonaute and GW182 proteins: an effective alliance in gene silencing. Biochem Soc Trans 41:855–860CrossRefPubMedGoogle Scholar
  14. 14.
    Jakymiw A et al (2005) Disruption of GW bodies impairs mammalian RNA interference. Nat Cell Biol 7:1267–1274CrossRefPubMedGoogle Scholar
  15. 15.
    Meister G et al (2005) Identification of novel argonaute-associated proteins. Curr Biol 15:2149–2155CrossRefPubMedGoogle Scholar
  16. 16.
    Liu J et al (2005) A role for the P-body component GW182 in microRNA function. Nat Cell Biol 7:1161–1166CrossRefGoogle Scholar
  17. 17.
    Rehwinkel J et al (2005) A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing. RNA 11:1640–1647CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Behm-Ansmant I et al (2006) mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes Dev 20:1885–1898CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Chekulaeva M et al (2011) miRNA repression involves GW182-mediated recruitment of CCR4-NOT through conserved W-containing motifs. Nat Struct Mol Biol 18:1218–1226CrossRefPubMedGoogle Scholar
  20. 20.
    Chen Y et al (2014) A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing. Mol Cell 54:737–750CrossRefPubMedGoogle Scholar
  21. 21.
    Mathys H et al (2014) Structural and biochemical insights to the role of the CCR4-NOT complex and DDX6 ATPase in microRNA repression. Mol Cell 54:751–765CrossRefPubMedGoogle Scholar
  22. 22.
    Braun JE, Huntzinger E, Izaurralde E (2013) The role of GW182 proteins in miRNA-mediated gene silencing. Adv Exp Med Biol 768:147–163CrossRefPubMedGoogle Scholar
  23. 23.
    Pfaff J et al (2013) Structural features of Argonaute-GW182 protein interactions. Proc Natl Acad Sci U S A 110:E3770–E3779CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Schirle NT, MacRae IJ (2012) The crystal structure of human Argonaute2. Science 336:1037–1040CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Baillat D, Shiekhattar R (2009) Functional dissection of the human TNRC6 (GW182-related) family of proteins. Mol Cell Biol 29:4144–4155CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Lazzaretti D, Tournier I, Izaurralde E (2009) The C-terminal domains of human TNRC6A, TNRC6B, and TNRC6C silence bound transcripts independently of Argonaute proteins. RNA 15:1059–1066CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Hauptmann J et al (2015) Biochemical isolation of Argonaute protein complexes by Ago-APP. Proc Natl Acad Sci U S A 112:11841–11845CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    El-Shami M et al (2007) Reiterated WG/GW motifs form functionally and evolutionarily conserved ARGONAUTE-binding platforms in RNAi-related components. Genes Dev 21:2539–2544CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Till S et al (2007) A conserved motif in Argonaute-interacting proteins mediates functional interactions through the Argonaute PIWI domain. Nat Struct Mol Biol 14:897–903CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.University of RegensburgRegensburgGermany

Personalised recommendations