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

  1. Greg S. Parker,
  2. Debra M. Eckert, and
  3. Brenda L. Bass
  1. Department of Biochemistry/HHMI, University of Utah, Salt Lake City, Utah 84112-5650, USA

Abstract

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.

Keywords

Footnotes

  • Reprint requests to: Brenda L. Bass, Department of Biochemistry/HHMI, University of Utah, 15 N. Medical Drive East, Rm. 4100, Salt Lake City, UT 84112-5650, USA; e-mail: bbass{at}biochem.utah.edu; fax: (801) 581-5379.

  • Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.2338706.

    • Received December 20, 2005.
    • Accepted February 20, 2006.
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