Molecular basis of DRB4-assisted long RNA processing and 21-nucleotide siRNA biogenesis by DCL4 in plants

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 substrates^2,3. In Arabidopsis, DICER-LIKE 4 (DCL4) produces 21-nucleotide siRNA in association with Double-Stranded RNA-Binding Protein 4 (DRB4) for post-transcriptional gene silencing^4-11. 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.