RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA
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.
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Footnotes
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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.
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Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.2338706.
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- Received December 20, 2005.
- Accepted February 20, 2006.
- Copyright © 2006 RNA Society