In Vitro RNA Cleavage Assay for Argonaute-family Proteins

Methods Mol Biol. 2008;442:29-43. doi: 10.1007/978-1-59745-191-8_3.

Abstract

Recent studies have revealed that Argonaute proteins are crucial components of the RNA-induced silencing complexes (RISCs) that direct both small interfering RNA (siRNA)- and microRNA (miRNA)-mediated gene silencing. Full complementarity between the small RNA and its target messenger RNA (mRNA) results in RISC-mediated cleavage ("Slicing") of the target mRNA. A subset of Argonaute proteins directly contributes to the target cleavage ("Slicer") activity of the RISC. We describe (in vitro) Slicer assays using endogenous Argonaute protein immunopurified from animal cells and recombinant Argonaute protein produced in and purified from Escherichia coli.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Argonaute Proteins
  • Drosophila / genetics
  • Drosophila / metabolism
  • Drosophila Proteins / genetics
  • Drosophila Proteins / isolation & purification
  • Drosophila Proteins / metabolism*
  • Eukaryotic Initiation Factor-2 / genetics
  • Eukaryotic Initiation Factor-2 / isolation & purification
  • Eukaryotic Initiation Factor-2 / metabolism*
  • Eukaryotic Initiation Factors
  • Gene Silencing
  • Humans
  • MicroRNAs / genetics
  • MicroRNAs / metabolism
  • RNA / genetics
  • RNA / metabolism*
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • RNA-Induced Silencing Complex / chemistry
  • RNA-Induced Silencing Complex / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • AGO1 protein, Drosophila
  • AGO2 protein, human
  • Argonaute Proteins
  • Drosophila Proteins
  • Eukaryotic Initiation Factor-2
  • Eukaryotic Initiation Factors
  • MicroRNAs
  • RNA, Small Interfering
  • RNA-Induced Silencing Complex
  • Recombinant Fusion Proteins
  • RNA