Small RNAs, including microRNA, small interfering RNA (siRNA) and PIWI-interacting RNA, are regulatory RNAs that play critical roles in gene regulation, development, viral defence and environmental response1. The biogenesis of microRNA and siRNA relies on the Dicer family ribonucleases to capture, measure and cleave their double-stranded RNA substrates2,3. In Arabidopsis, DICER-LIKE 4 (DCL4) produces 21-nucleotide siRNA in association with Double-Stranded RNA-Binding Protein 4 (DRB4) for post-transcriptional gene silencing4–11. Here we determined the structures of the DCL4–RNA complex in a dicing-competent conformation and the DCL4–DRB4–RNA complex in a pre-dicing conformation. DCL4 measures 21 nucleotides along RNA between its PAZ and RNase III domains to determine the product siRNA length. A DCL4-specific loop locates the second double-stranded RNA binding domain of DCL4 and DRB4 to a distal position of the substrate RNA, yielding a preference for long RNA substrates. Our studies demonstrate the molecular basis of substrate recognition, length measurement and long RNA preference by the DCL4–DRB4 complex for 21-nucleotide siRNA biogenesis in plants.
