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Reconstitution of RNA Interference Machinery

  • Shintaro Iwasaki
  • Yukihide TomariEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

siRNA miRNA Chaperone Argonaute 

Notes

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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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Institute of Molecular and Cellular BiosciencesThe University of TokyoTokyoJapan
  2. 2.RNA Systems Biochemistry LaboratoryRIKENWakoJapan

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