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Methods Mol Biol. 2018;1680:131-143. doi: 10.1007/978-1-4939-7339-2_9.

Reconstitution of RNA Interference Machinery.

Author information

1
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0032, Japan.
2
RNA Systems Biochemistry Laboratory, RIKEN, Wako, Saitama, 351-0198, Japan.
3
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0032, Japan. tomari@iam.u-tokyo.ac.jp.

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.

KEYWORDS:

Argonaute; Chaperone; miRNA; siRNA

PMID:
29030846
DOI:
10.1007/978-1-4939-7339-2_9
[Indexed for MEDLINE]

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