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 RIS Download
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