Advertisement

Single-Molecule Analysis for RISC Assembly and Target Cleavage

  • Hiroshi M. Sasaki
  • Hisashi Tadakuma
  • Yukihide Tomari
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1680)

Abstract

RNA-induced silencing complex (RISC) is a small RNA–protein complex that mediates silencing of complementary target RNAs. Biochemistry has been successfully used to characterize the molecular mechanism of RISC assembly and function for nearly two decades. However, further dissection of intermediate states during the reactions has been warranted to fill in the gaps in our understanding of RNA silencing mechanisms. Single-molecule analysis with total internal reflection fluorescence (TIRF) microscopy is a powerful imaging-based approach to interrogate complex formation and dynamics at the individual molecule level with high sensitivity. Combining this technique with our recently established in vitro reconstitution system of fly Ago2-RISC, we have developed a single-molecule observation system for RISC assembly. In this chapter, we summarize the detailed protocol for single-molecule analysis of chaperone-assisted assembly of fly Ago2-RISC as well as its target cleavage reaction.

Key words

Single-molecule imaging RNA interference Argonaute RISC Small interfering RNA Total internal reflection fluorescence microscopy 

Notes

Acknowledgements

We thank S. Iwasaki for constructing vectors for protein expression and establish the in vitro reconstitution system of fly Ago2-RISC assembly. This work was supported in part by Grants-in-Aid for Scientific Research on Innovative Areas (‘Functional machinery for non-coding RNAs’ 21115002 and ‘Non-coding RNA neo-taxonomy’ 26113007) (to H.T. and Y.T.), and Grants-in-Aid for challenging Exploratory Research (24657115 and 26650047) (to H.M.S.) from The Ministry of Education, Culture, Sports, Science and Technology in Japan.

References

  1. 1.
    Carthew RW, Sontheimer EJ (2009) Origins and mechanisms of miRNAs and siRNAs. Cell 136(4):642–655CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Kawamata T, Tomari Y (2010) Making Risc. Trends Biochem Sci 35(7):368–376CrossRefPubMedGoogle Scholar
  3. 3.
    Liu Q, Rand TA, Kalidas S, Du F, Kim HE, Smith DP, Wang X (2003) R2d2, a bridge between the initiation and effector steps of the Drosophila Rnai pathway. Science 301(5641):1921–1925CrossRefPubMedGoogle Scholar
  4. 4.
    Pham JW, Pellino JL, Lee YS, Carthew RW, Sontheimer EJ (2004) A dicer-2-dependent 80s complex cleaves targeted Mrnas during Rnai in Drosophila. Cell 117(1):83–94CrossRefPubMedGoogle Scholar
  5. 5.
    Tomari Y, Matranga C, Haley B, Martinez N, Zamore PD (2004) A protein sensor for siRNA asymmetry. Science 306(5700):1377–1380CrossRefPubMedGoogle Scholar
  6. 6.
    Tomari Y, Du T, Haley B, Schwarz DS, Bennett R, Cook HA, Koppetsch BS, Theurkauf WE, Zamore PD (2004) Risc assembly defects in the drosophila Rnai mutant Armitage. Cell 116(6):831–841CrossRefPubMedGoogle Scholar
  7. 7.
    Iki T, Yoshikawa M, Nishikiori M, Jaudal MC, Matsumoto-Yokoyama E, Mitsuhara I, Meshi T, Ishikawa M (2010) In vitro assembly of plant RNA-induced silencing complexes facilitated by molecular chaperone Hsp90. Mol Cell 39(2):282–291CrossRefPubMedGoogle Scholar
  8. 8.
    Iwasaki S, Kobayashi M, Yoda M, Sakaguchi Y, Katsuma S, Suzuki T, Tomari Y (2010) Hsc70/hsp90 chaperone machinery mediates Atp-dependent Risc loading of small RNA duplexes. Mol Cell 39(2):292–299CrossRefPubMedGoogle Scholar
  9. 9.
    Kawamata T, Seitz H, Tomari Y (2009) Structural determinants of Mirnas for Risc loading and slicer-independent unwinding. Nat Struct Mol Biol 16(9):953–960CrossRefPubMedGoogle Scholar
  10. 10.
    Yoda M, Kawamata T, Paroo Z, Ye X, Iwasaki S, Liu Q, Tomari Y (2010) Atp-dependent human Risc assembly pathways. Nat Struct Mol Biol 17(1):17–23CrossRefPubMedGoogle Scholar
  11. 11.
    Miyoshi T, Takeuchi A, Siomi H, Siomi MC (2010) A direct role for Hsp90 in pre-Risc formation in drosophila. Nat Struct Mol Biol 17(8):1024–1026CrossRefPubMedGoogle Scholar
  12. 12.
    Betancur JG, Tomari Y (2012) Dicer is dispensable for asymmetric Risc loading in mammals. RNA 18(1):24–30CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Kawamata T, Yoda M, Tomari Y (2011) Multilayer checkpoints for Microrna authenticity during Risc assembly. EMBO Rep 12(9):944–949CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Kawamata T, Tomari Y (2011) Native gel analysis for Risc assembly. Methods Mol Biol 725:91–105CrossRefPubMedGoogle Scholar
  15. 15.
    Iwasaki S, Sasaki HM, Sakaguchi Y, Suzuki T, Tadakuma H, Tomari Y (2015) Defining fundamental steps in the assembly of the drosophila Rnai enzyme complex. Nature 521(7553):533–536CrossRefPubMedGoogle Scholar
  16. 16.
    Yao C, Sasaki HM, Ueda T, Tomari Y, Tadakuma H (2015) Single-molecule analysis of the target cleavage reaction by the drosophila Rnai enzyme complex. Mol Cell 59(1):125–132CrossRefPubMedGoogle Scholar
  17. 17.
    Jo MH, Shin S, Jung SR, Kim E, Song JJ, Hohng S (2015) Human Argonaute 2 has diverse reaction pathways on target Rnas. Mol Cell 59(1):117–124CrossRefPubMedGoogle Scholar
  18. 18.
    Salomon WE, Jolly SM, Moore MJ, Zamore PD, Serebrov V (2015) Single-molecule imaging reveals that Argonaute reshapes the binding properties of its nucleic acid guides. Cell 162(1):84–95CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Miyazono Y, Hayashi M, Karagiannis P, Harada Y, Tadakuma H (2010) Strain through the neck linker ensures Processive runs: a DNA-Kinesin hybrid Nanomachine study. EMBO J 29(1):93–106CrossRefPubMedGoogle Scholar
  20. 20.
    Yildiz A, Forkey JN, Mckinney SA, Ha T, Goldman YE, Selvin PR (2003) Myosin V walks hand-over-hand: single Fluorophore imaging with 1.5-nm localization. Science 300(5628):2061–2065CrossRefPubMedGoogle Scholar
  21. 21.
    Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82(5):2775–2783CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Zhou ZP, Shimizu Y, Tadakuma H, Taguchi H, Ito K, Ueda T (2011) Single molecule imaging of the trans-translation entry process via anchoring of the tagged ribosome. J Biochem 149(5):609–618CrossRefPubMedGoogle Scholar
  23. 23.
    Tseng Q, Duchemin-Pelletier E, Deshiere A, Balland M, Guillou H, Filhol O, Thery M (2012) Spatial organization of the extracellular matrix regulates cell-cell junction positioning. Proc Natl Acad Sci U S A 109(5):1506–1511CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Ueno T, Taguchi H, Tadakuma H, Yoshida M, Funatsu T (2004) GroEL mediates protein folding with a two successive timer mechanism. Mol Cell 14:423–434CrossRefPubMedGoogle Scholar
  25. 25.
    Small A, Stahlheber S (2014) Fluorophore localization algorithms for super-resolution microscopy. Nat Methods 11(3):267–279CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Hiroshi M. Sasaki
    • 1
    • 2
  • Hisashi Tadakuma
    • 3
    • 4
  • Yukihide Tomari
    • 1
    • 3
  1. 1.Intsitute for Molecular and Cellular BiosciencesThe University of TokyoTokyoJapan
  2. 2.Wyss Institute for Biologically Inspired EngineeringHarvard UniversityBostonUSA
  3. 3.Department of Computational Biology and Medical Sciences, Graduate School of Frontier SciencesThe University of TokyoTokyoJapan
  4. 4.Institute for Protein ResearchOsaka UniversityOsakaJapan

Personalised recommendations