Generation of catalytic human Ago4 identifies structural elements important for RNA cleavage

RNA. 2014 Oct;20(10):1532-8. doi: 10.1261/rna.045203.114. Epub 2014 Aug 11.


Argonaute proteins bind small RNAs and mediate cleavage of complementary target RNAs. The human Argonaute protein Ago4 is catalytically inactive, although it is highly similar to catalytic Ago2. Here, we have generated Ago2-Ago4 chimeras and analyzed their cleavage activity in vitro. We identify several specific features that inactivate Ago4: the catalytic center, short sequence elements in the N-terminal domain, and an Ago4-specific insertion in the catalytic domain. In addition, we show that Ago2-mediated cleavage of the noncanonical miR-451 precursor can be carried out by any catalytic human Ago protein. Finally, phylogenetic analyses establish evolutionary distances between the Ago proteins. Interestingly, these distances do not fully correlate with the structural changes inactivating them, suggesting functional adaptations of individual human Ago proteins.

Keywords: Ago4; Argonaute proteins; RNAi; gene silencing; microRNAs; siRNAs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Argonaute Proteins / chemistry*
  • Argonaute Proteins / genetics*
  • Argonaute Proteins / metabolism
  • Blotting, Northern
  • Catalysis
  • Eukaryotic Initiation Factors / chemistry*
  • Eukaryotic Initiation Factors / genetics*
  • Eukaryotic Initiation Factors / metabolism
  • HEK293 Cells
  • Humans
  • MicroRNAs / genetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Phylogeny
  • Protein Conformation
  • RNA Cleavage / genetics*
  • RNA, Small Interfering / genetics
  • Sequence Homology, Amino Acid


  • AGO2 protein, human
  • AGO4 protein, human
  • Argonaute Proteins
  • Eukaryotic Initiation Factors
  • MicroRNAs
  • RNA, Small Interfering