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Argonaute Proteins pp 131–143Cite as

Reconstitution of RNA Interference Machinery

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1680))

Abstract

Small RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs), silence protein expression from target mRNAs bearing their complementary sequences, via the formation of the effector complex called RNA-induced silencing complex (RISC). Although the mechanism of RISC assembly has been studied for nearly two decades, the detailed mechanism has still remained unclear in part due to the lack of a pure reconstitution system. Recently, we identified all the core proteins necessary for RISC assembly in flies and successfully recapitulated the assembly of catalytically active RISC with eight recombinant proteins. The reconstitution system provides a versatile framework for detailed studies of RISC assembly, including single molecule analysis as described in another chapter in this issue.

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Acknowledgments

We are grateful to members of Tomari laboratory and Iwasaki laboratory for critical reading of this manuscript. This work is supported by 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 Y.T.), and a Grant-in-Aid for Scientific Research on Innovative Areas (‘nascent chain biology’ JP17H05679) and Grant-in-Aid for Young Scientists (‘Start-up’ 23870004 and ‘A’ JP17H04998) (to S.I.) from The Ministry of Education, Culture, Sports, Science and Technology in Japan.

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Authors and Affiliations

  1. Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0032, Japan

    Shintaro Iwasaki & Yukihide Tomari

  2. RNA Systems Biochemistry Laboratory, RIKEN, Wako, Saitama, 351-0198, Japan

    Shintaro Iwasaki

Authors
  1. Shintaro Iwasaki
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  2. Yukihide Tomari
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Corresponding author

Correspondence to Yukihide Tomari .

Editor information

Editors and Affiliations

  1. Temasek Lifesciences Laboratory, National University of Singapore, Singapore, Singapore

    Katsutomo Okamura

  2. Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio, USA

    Kotaro Nakanishi

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Iwasaki, S., Tomari, Y. (2018). Reconstitution of RNA Interference Machinery. In: Okamura, K., Nakanishi, K. (eds) Argonaute Proteins. Methods in Molecular Biology, vol 1680. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7339-2_9

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  • DOI: https://doi.org/10.1007/978-1-4939-7339-2_9

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7338-5

  • Online ISBN: 978-1-4939-7339-2

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