Native gel analysis for RISC assembly

Methods Mol Biol. 2011;725:91-105. doi: 10.1007/978-1-61779-046-1_7.

Abstract

Small-interfering RNAs (siRNAs) and microRNAs (miRNAs) regulate expression of their target mRNAs via the RNA-induced silencing complex (RISC). A core component of RISC is the Argonaute (Ago) protein, which dictates the RISC function. In Drosophila, miRNAs and siRNAs are generally loaded into Ago1-containing RISC (Ago1-RISC) and Ago2-containing RISC (Ago2-RISC), respectively. We developed a native agarose gel system to directly detect Ago1-RISC, Ago2-RISC, and their precursor complexes. Methods presented here will provide powerful tools to biochemically dissect the RISC assembly pathways.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Argonaute Proteins
  • Drosophila / genetics
  • Drosophila / metabolism
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism
  • Electrophoresis, Agar Gel*
  • Eukaryotic Initiation Factors / metabolism
  • Isotope Labeling
  • Mutation / genetics
  • RNA / isolation & purification
  • RNA / metabolism
  • RNA Helicases / genetics
  • RNA-Induced Silencing Complex / metabolism*
  • Ribonuclease III / genetics

Substances

  • AGO1 protein, Drosophila
  • Argonaute Proteins
  • Drosophila Proteins
  • Eukaryotic Initiation Factors
  • RNA-Induced Silencing Complex
  • RNA
  • DCR-2 protein, Drosophila
  • Ribonuclease III
  • RNA Helicases