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Peptide-Based Inhibition of miRNA-Guided Gene Silencing

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Drug Target miRNA

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1517))

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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References

  1. Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136:215–233

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Carthew RW, Sontheimer EJ (2009) Origins and mechanisms of miRNAs and siRNAs. Cell 136:642–655

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Dueck A, Meister G (2014) Assembly and function of small RNA – argonaute protein complexes. Biol Chem 395:611–629

    Article  CAS  PubMed  Google Scholar 

  4. Ipsaro JJ, Joshua-Tor L (2015) From guide to target: molecular insights into eukaryotic RNA-interference machinery. Nat Struct Mol Biol 22:20–28

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Jinek M, Doudna JA (2009) A three-dimensional view of the molecular machinery of RNA interference. Nature 457:405–412

    Article  CAS  PubMed  Google Scholar 

  6. Kwak PB, Tomari Y (2012) The N domain of argonaute drives duplex unwinding during RISC assembly. Nat Struct Mol Biol 19:145–151

    Article  CAS  PubMed  Google Scholar 

  7. Song JJ, Liu J, Tolia NH, Schneiderman J, Smith SK, Martienssen RA, Hannon GJ, Joshua-Tor L (2003) The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes. Nat Struct Biol 10:1026–1032

    Article  CAS  PubMed  Google Scholar 

  8. Ma JB, Ye K, Patel DJ (2004) Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain. Nature 429:318–322

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lingel A, Simon B, Izaurralde E, Sattler M (2003) Structure and nucleic-acid binding of the Drosophila argonaute 2 PAZ domain. Nature 426:465–469

    Article  CAS  PubMed  Google Scholar 

  10. Yan KS, Yan S, Farooq A, Han A, Zeng L, Zhou MM (2003) Structure and conserved RNA binding of the PAZ domain. Nature 426:468–474

    Article  PubMed  Google Scholar 

  11. Ma JB, Yuan YR, Meister G, Pei Y, Tuschl T, Patel DJ (2005) Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature 434:666–670

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Parker JS, Roe SM, Barford D (2005) Structural insights into mRNA recognition from a PIWI domain-siRNA guide complex. Nature 434:663–666

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Song JJ, Smith SK, Hannon GJ, Joshua-Tor L (2004) Crystal structure of argonaute and its implications for RISC slicer activity. Science 305:1434–1437

    Article  CAS  PubMed  Google Scholar 

  14. Yuan YR, Pei Y, Ma JB, Kuryavyi V, Zhadina M, Meister G, Chen HY, Dauter Z, Tuschl T, Patel DJ (2005) Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage. Mol Cell 19:405–419

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Wang B, Li S, Qi HH, Chowdhury D, Shi Y, Novina CD (2009) Distinct passenger strand and mRNA cleavage activities of human argonaute proteins. Nat Struct Mol Biol 16:1259–1266

    Article  CAS  PubMed  Google Scholar 

  16. Meister G, Landthaler M, Patkaniowska A, Dorsett Y, Teng G, Tuschl T (2004) Human argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol Cell 15:185–197

    Article  CAS  PubMed  Google Scholar 

  17. Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ, Hammond SM, Joshua-Tor L, Hannon GJ (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305:1437–1441

    Article  CAS  PubMed  Google Scholar 

  18. Elkayam E, Kuhn CD, Tocilj A, Haase AD, Greene EM, Hannon GJ, Joshua-Tor L (2012) The structure of human argonaute-2 in complex with miR-20a. Cell 150:100–110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Schirle NT, MacRae IJ (2012) The crystal structure of human argonaute2. Science 336:1037–1040

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Tuschl T, Zamore PD, Lehmann R, Bartel DP, Sharp PA (1999) Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 13:3191–3197

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101:25–33

    Article  CAS  PubMed  Google Scholar 

  22. Hammond SM, Bernstein E, Beach D, Hannon GJ (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404:293–296

    Article  CAS  PubMed  Google Scholar 

  23. Chen PY, Meister G (2005) microRNA-guided posttranscriptional gene regulation. Biol Chem 386:1205–1218

    Article  CAS  PubMed  Google Scholar 

  24. Liu J, Rivas FV, Wohlschlegel J, Yates JR 3rd, Parker R, Hannon GJ (2005) A role for the P-body component GW182 in microRNA function. Nat Cell Biol 7:1161–1166

    Article  CAS  Google Scholar 

  25. Rehwinkel J, Behm-Ansmant I, Gatfield D, Izaurralde E (2005) A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing. RNA 11:1640–1647

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Meister G, Landthaler M, Peters L, Chen PY, Urlaub H, Luhrmann R, Tuschl T (2005) Identification of novel argonaute-associated proteins. Curr Biol 15:2149–2155

    Article  CAS  PubMed  Google Scholar 

  27. Liu J, Valencia-Sanchez MA, Hannon GJ, Parker R (2005) MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies. Nat Cell Biol 7:719–723

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Sen GL, Blau HM (2005) Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies. Nat Cell Biol 7:633–636

    Article  CAS  PubMed  Google Scholar 

  29. Jakymiw A, Lian S, Eystathioy T, Li S, Satoh M, Hamel JC, Fritzler MJ, Chan EK (2005) Disruption of GW bodies impairs mammalian RNA interference. Nat Cell Biol 7:1267–1274

    Article  PubMed  Google Scholar 

  30. Jonas S, Izaurralde E (2015) Towards a molecular understanding of microRNA-mediated gene silencing. Nat Rev Genet 16:421–433

    Article  CAS  PubMed  Google Scholar 

  31. Chen Y, Boland A, Kuzuoglu-Ozturk D, Bawankar P, Loh B, Chang CT, Weichenrieder O, Izaurralde E (2014) A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing. Mol Cell 54:737–750

    Article  CAS  PubMed  Google Scholar 

  32. Mathys H, Basquin J, Ozgur S, Czarnocki-Cieciura M, Bonneau F, Aartse A, Dziembowski A, Nowotny M, Conti E, Filipowicz W (2014) Structural and biochemical insights to the role of the CCR4-NOT complex and DDX6 ATPase in MicroRNA repression. Mol Cell 54:751–765

    Article  CAS  PubMed  Google Scholar 

  33. Huntzinger E, Izaurralde E (2011) Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nat Rev Genet 12:99–110

    Article  CAS  PubMed  Google Scholar 

  34. Pfaff J, Hennig J, Herzog F, Aebersold R, Sattler M, Niessing D, Meister G (2013) Structural features of argonaute-GW182 protein interactions. Proc Natl Acad Sci U S A 110:E3770–E3779

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Calin GA, Croce CM (2006) MicroRNA signatures in human cancers. Nat Rev Cancer 6:857–866

    Article  CAS  PubMed  Google Scholar 

  36. Kasinski AL, Slack FJ (2011) Epigenetics and genetics. MicroRNAs en route to the clinic: progress in validating and targeting microRNAs for cancer therapy. Nat Rev Cancer 11:849–864

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Chen K, Rajewsky N (2007) The evolution of gene regulation by transcription factors and microRNAs. Nat Rev Genet 8:93–103

    Article  CAS  PubMed  Google Scholar 

  38. Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120:15–20

    Article  CAS  PubMed  Google Scholar 

  39. Till S, Lejeune E, Thermann R, Bortfeld M, Hothorn M, Enderle D, Heinrich C, Hentze MW, Ladurner AG (2007) A conserved motif in argonaute-interacting proteins mediates functional interactions through the Argonaute PIWI domain. Nat Struct Mol Biol 14:897–903

    Article  CAS  PubMed  Google Scholar 

  40. Hauptmann J, Schraivogel D, Bruckmann A, Manickavel S, Jakob L, Eichner N, Pfaff J, Urban M, Sprunck S, Hafner M et al (2015) Biochemical isolation of argonaute protein complexes by Ago-APP. Proc Natl Acad Sci U S A 112:11841–11845

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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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).

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Authors and Affiliations

  1. Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Universitätsstraße 31, 93053, Regensburg, Germany

    Johannes Danner, Balagopal Pai, Ludwig Wankerl & Gunter Meister

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  1. Johannes Danner
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  2. Balagopal Pai
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  3. Ludwig Wankerl
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  4. Gunter Meister
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Corresponding author

Correspondence to Gunter Meister .

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Editors and Affiliations

  1. BEROCEUTICA GmbH, Potsdam, Germany

    Marco F. Schmidt

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Danner, J., Pai, B., Wankerl, L., Meister, G. (2017). Peptide-Based Inhibition of miRNA-Guided Gene Silencing. In: Schmidt, M. (eds) Drug Target miRNA. Methods in Molecular Biology, vol 1517. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6563-2_14

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  • DOI: https://doi.org/10.1007/978-1-4939-6563-2_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6561-8

  • Online ISBN: 978-1-4939-6563-2

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