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Kinetic Analysis of Target RNA Binding and Slicing by Human Argonaute 2 Protein

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Drug Target miRNA

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

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Abstract

Analyzing the mechanisms of Argonaute-mediated gene silencing is essential to the understanding of RNA interference (RNAi). RNAi is a process to regulate gene expression on a posttranscriptional level. Directed by single-stranded small RNA guides, Argonaute 2 binds complementary target RNAs, and if the guide displays full complementarity to the targeted sequence, Argonaute 2 slices the bound target RNA. This on the one hand is an important mechanism to regulate gene expression in the cell and on the other hand represents a powerful tool to interfere with harmful gene expression levels. Here, we present techniques to kinetically characterize recombinant Argonaute 2-mediated guide and target binding as well as target RNA slicing. We focus on fluorescence-based steady-state and in particular pre-steady-state techniques to unravel mechanistic details. Furthermore, we describe a cleavage assay to analyze Argonaute 2-mediated slicing using radioactively labeled target strands.

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References

  1. Fire A, Xu SQ, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811. doi:10.1038/35888

    Article  CAS  PubMed  Google Scholar 

  2. 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. doi:10.1016/j.molcel.2004.07.007

    Article  CAS  PubMed  Google Scholar 

  3. Liu J, Carmell M, Rivas FV, Marsden CG, Thomson JM, Song J-J et al (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305:1437–1441. doi:10.1126/science.1102513

    Article  CAS  PubMed  Google Scholar 

  4. Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409:363–366. doi:10.1038/35053110

    Article  CAS  PubMed  Google Scholar 

  5. MacRae IJ, Zhou K, Li F, Repic A, Brooks AN, Cande WZ et al (2006) Structural basis for double-stranded RNA processing by Dicer. Science 311:195–198. doi:10.1126/science.1121638

    Article  CAS  PubMed  Google Scholar 

  6. Elbashir SM, Lendeckel W, Tuschl T (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15:188–200. doi:10.1101/gad.862301

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Maniataki E, Mourelatos Z (2005) A human, ATP-independent, RISC assembly machine fueled by pre-miRNA. Genes Dev 19:2979–2990. doi:10.1101/gad.1384005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. MacRae IJ, Ma E, Zhou M, Robinson CV, Doudna JA (2008) In vitro reconstitution of the human RISC-loading complex. Proc Natl Acad Sci U S A 105:512–517. doi:10.1073/pnas.0710869105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hutvagner G, Simard MJ (2008) Argonaute proteins: key players in RNA silencing. Nat Rev Mol Cell Biol 9:22–32. doi:10.1038/nrm2321

    Article  CAS  PubMed  Google Scholar 

  10. Leuschner PJ, Ameres SL, Kueng S, Martinez J (2006) Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep 7:314–320. doi:10.1038/sj.embor.7400637

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Rand TA, Petersen S, Du F, Wang X (2005) Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation. Cell 123:621–629. doi:10.1016/j.cell.2005.10.020

    Article  CAS  PubMed  Google Scholar 

  12. Matranga C, Tomari Y, Shin C, Bartel DP, Zamore PD (2005) Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123:607–620. doi:10.1016/j.cell.2005.08.044

    Article  CAS  PubMed  Google Scholar 

  13. Kim K, Lee YS, Carthew RW (2007) Conversion of pre-RISC to holo-RISC by Ago2 during assembly of RNAi complexes. RNA 13:22–29. doi:10.1261/rna.283207

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Siomi H, Siomi MC (2009) On the road to reading the RNA-interference code. Nature 457:396–404. doi:10.1038/nature07754

    Article  CAS  PubMed  Google Scholar 

  15. Willkomm S, Restle T (2015) Conformational dynamics of Ago-mediated silencing processes. Int J Mol Sci 16:14769–14785. doi:10.3390/ijms160714769

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Dornseifer S, Willkomm S, Kretschmer-Kazemi Far R, Liebschwager J, Beltsiou F, Frank K et al (2015) RNAi revised – target mRNA-dependent enhancement of gene silencing. Nucleic Acids Res 43:10623–10632. doi:10.1093/nar/gkv1200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Deerberg A, Willkomm S, Restle T (2013) Minimal mechanistic model of siRNA-dependent target RNA slicing by recombinant human Argonaute 2 protein. Proc Natl Acad Sci U S A 110:17850–17855. doi:10.1073/pnas.1217838110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Fersht A (1985) Enzyme structure and mechanism, 2nd edn. W.H. Freeman & Co Ltd., New York, NY

    Google Scholar 

  19. Gutfreund H (1995) Kinetics for the life sciences: receptors, transmitters and catalysts. Cambridge University Press, Cambridge

    Book  Google Scholar 

  20. Johnson KA (1992) Transient-state kinetic analysis of enzyme reaction pathways. Enzymes 20:1–61. doi:10.1016/S1874-6047(08)60019-0

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Institute of Molecular Medicine, Universitätsklinikum Schleswig-Holstein, University of Lübeck, Ratzeburger Allee 160, 23538, Lübeck, Germany

    Sarah Willkomm & Tobias Restle

  2. Department of Microbiology, Single Molecule Biochemistry Lab, Universität Regensburg, Regensburg, Germany

    Sarah Willkomm

Authors
  1. Sarah Willkomm
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  2. Tobias Restle
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Corresponding author

Correspondence to Tobias Restle .

Editor information

Editors and Affiliations

  1. BEROCEUTICA GmbH, Potsdam, Germany

    Marco F. Schmidt

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Willkomm, S., Restle, T. (2017). Kinetic Analysis of Target RNA Binding and Slicing by Human Argonaute 2 Protein. 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_19

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  • DOI: https://doi.org/10.1007/978-1-4939-6563-2_19

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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

  • eBook Packages: Springer Protocols

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