Reconstitution of RNA Interference Machinery

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

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.

MeSH terms

  • Animals
  • Argonaute Proteins / metabolism
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism
  • Gene Expression
  • Gene Silencing
  • HSC70 Heat-Shock Proteins / metabolism
  • Janus Kinases / metabolism
  • MicroRNAs / genetics*
  • RNA Interference*
  • RNA, Small Interfering / genetics*
  • RNA-Induced Silencing Complex
  • Recombinant Proteins / metabolism
  • Ribonuclease III / metabolism
  • Transcription Factors / metabolism

Substances

  • Argonaute Proteins
  • Drosophila Proteins
  • HSC70 Heat-Shock Proteins
  • Hsc70-4 protein, Drosophila
  • MicroRNAs
  • RNA, Small Interfering
  • RNA-Induced Silencing Complex
  • Recombinant Proteins
  • Transcription Factors
  • Janus Kinases
  • hop protein, Drosophila
  • Ribonuclease III