Biochemical identification of Argonaute 2 as the sole protein required for RNA-induced silencing complex activity

Proc Natl Acad Sci U S A. 2004 Oct 5;101(40):14385-9. doi: 10.1073/pnas.0405913101. Epub 2004 Sep 27.

Abstract

RNA interference is carried out by the small double-stranded RNA-induced silencing complex (RISC). The RISC-bound small RNA guides the RISC complex to identify and cleave mRNAs with complementary sequences. The proteins that make up the RISC complex and cleave mRNA have not been unequivocally defined. Here, we report the biochemical purification of RISC activity to homogeneity from Drosophila Schnieder 2 cell extracts. Argonaute 2 (Ago-2) is the sole protein component present in the purified, functional RISC. By using a bioinformatics method that combines sequence-profile analysis with predicted protein secondary structure, we found homology between the PIWI domain of Ago-2 and endonuclease V and identified potential active-site amino acid residues within the PIWI domain of Ago-2.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Argonaute Proteins
  • Cell Line
  • Deoxyribonuclease (Pyrimidine Dimer) / chemistry
  • Deoxyribonuclease (Pyrimidine Dimer) / genetics
  • Drosophila / genetics
  • Drosophila / metabolism
  • Drosophila Proteins / chemistry
  • Drosophila Proteins / genetics
  • Drosophila Proteins / isolation & purification*
  • Molecular Sequence Data
  • Proteins / chemistry
  • Proteins / genetics
  • Proteins / isolation & purification
  • RNA Interference*
  • RNA, Small Interfering / genetics
  • RNA-Induced Silencing Complex / chemistry
  • RNA-Induced Silencing Complex / genetics
  • RNA-Induced Silencing Complex / isolation & purification*
  • Sequence Homology, Amino Acid
  • Sodium Chloride

Substances

  • AGO2 protein, Drosophila
  • Argonaute Proteins
  • Drosophila Proteins
  • Proteins
  • RNA, Small Interfering
  • RNA-Induced Silencing Complex
  • piwi protein, Drosophila
  • Sodium Chloride
  • Deoxyribonuclease (Pyrimidine Dimer)