Substrate-specific kinetics of Dicer-catalyzed RNA processing

J Mol Biol. 2010 Dec 3;404(3):392-402. doi: 10.1016/j.jmb.2010.09.030. Epub 2010 Oct 13.


The specialized ribonuclease Dicer plays a central role in eukaryotic gene expression by producing small regulatory RNAs-microRNAs (miRNAs) and short interfering RNAs (siRNAs)-from larger double-stranded RNA (dsRNA) substrates. Although Dicer will cleave both imperfectly base-paired hairpin structures (pre-miRNAs) and perfect duplexes (pre-siRNAs) in vitro, it has not been clear whether these are mechanistically equivalent substrates and how dsRNA binding proteins such as trans-activation response (TAR) RNA binding protein (TRBP) influence substrate selection and RNA processing efficiency. We show here that human Dicer is much faster at processing a pre-miRNA substrate compared to a pre-siRNA substrate under both single and multiple turnover conditions. Maximal cleavage rates (V(max)) calculated by Michaelis-Menten analysis differed by more than 100-fold under multiple turnover conditions. TRBP was found to enhance dicing of both substrates to similar extents, and this stimulation required the two N-terminal dsRNA binding domains of TRBP. These results demonstrate that multiple factors influence dicing kinetics. While TRBP stimulates dicing by enhancing the stability of Dicer-substrate complexes, Dicer itself generates product RNAs at rates determined at least in part by the structural properties of the substrate.

MeSH terms

  • Amino Acid Substitution
  • Base Sequence
  • DEAD-box RNA Helicases / chemistry
  • DEAD-box RNA Helicases / genetics
  • DEAD-box RNA Helicases / metabolism*
  • Humans
  • In Vitro Techniques
  • Kinetics
  • MicroRNAs / genetics
  • MicroRNAs / metabolism
  • Models, Biological
  • Mutagenesis, Site-Directed
  • Phosphorylation
  • Protein Interaction Domains and Motifs
  • RNA Precursors / genetics
  • RNA Precursors / metabolism
  • RNA Processing, Post-Transcriptional*
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Ribonuclease III / chemistry
  • Ribonuclease III / genetics
  • Ribonuclease III / metabolism*
  • Substrate Specificity


  • MicroRNAs
  • RNA Precursors
  • RNA-Binding Proteins
  • Recombinant Proteins
  • mirnlet7 microRNA, human
  • trans-activation responsive RNA-binding protein
  • DICER1 protein, human
  • Ribonuclease III
  • DEAD-box RNA Helicases