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RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA.

In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not required in subsequent steps, and how RDE-4 distinguishes between long dsRNA and short siRNA is unclear. We report the first detailed analysis of RDE-4 binding, using purified recombinant RDE-4 and various truncated proteins. We find that, similar to other dsRBPs, RDE-4 is not sequence-specific. However, consistent with its in vivo roles, RDE-4 binds with higher affinity to long dsRNA. We also observe that RDE-4 is a homodimer in solution, and that the C-terminal domain of the protein is required for dimerization. Using extracts from wild-type and rde-4 mutant C. elegans, we show that the C-terminal dimerization domain is required for the production of siRNA. Our findings suggest a model for RDE-4 function during the initiation of RNAi.

Pubmed ID: 16603715

Authors

  • Parker GS
  • Eckert DM
  • Bass BL

Journal

RNA (New York, N.Y.)

Publication Data

May 27, 2006

Associated Grants

  • Agency: NIGMS NIH HHS, Id: GM067106
  • Agency: NIGMS NIH HHS, Id: GM08537
  • Agency: NIGMS NIH HHS, Id: R01 GM067106
  • Agency: NIGMS NIH HHS, Id: R01 GM067106-03

Mesh Terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Binding, Competitive
  • Caenorhabditis elegans
  • Caenorhabditis elegans Proteins
  • DEAD-box RNA Helicases
  • Dimerization
  • Electrophoresis, Polyacrylamide Gel
  • Electrophoretic Mobility Shift Assay
  • Embryo, Nonmammalian
  • Kinetics
  • Models, Biological
  • Molecular Sequence Data
  • Molecular Weight
  • Protein Structure, Tertiary
  • RNA Helicases
  • RNA Interference
  • RNA, Double-Stranded
  • RNA, Small Interfering
  • RNA-Binding Proteins