The C-terminal dsRNA-binding domain of Drosophila Dicer-2 is crucial for efficient and high-fidelity production of siRNA and loading of siRNA to Argonaute2
- ↵* Corresponding author; email: fukunaga{at}jhmi.edu
Abstract
Drosophila Dicer-2 efficiently and precisely produces 21-nt siRNAs from long double-stranded RNA (dsRNA) substrates and loads these siRNAs onto the effector protein Argonaute2 for RNA silencing. The functional roles of each domain of the multi-domain Dicer-2 enzyme in the production and loading of siRNAs are not fully understood. Here we characterized Dicer-2 mutants lacking either the N-terminal helicase domain or C-terminal dsRNA-binding domain (CdsRBD) (ΔHelicase and ΔCdsRBD, respectively) in vivo and in vitro. We found that ΔCdsRBD Dicer-2 produces siRNAs with lowered efficiency and length-fidelity, producing a smaller ratio of 21 nt siRNAs and higher ratios of 20 nt and 22 nt siRNAs in vivo and in vitro. We also found that ΔCdsRBD Dicer-2 cannot load siRNA duplexes to Argonaute2 in vitro. Consistent with these findings, we found that ΔCdsRBD Dicer-2 causes partial loss of RNA silencing activity in vivo. Thus, Dicer-2 CdsRBD is crucial for the efficiency and length fidelity in siRNA production and for siRNA loading. Together with our previously published findings, we propose that CdsRBD binds the proximal body region of a long dsRNA substrate whose 5′-monophosphate end is anchored by the phosphate-binding pocket in the PAZ domain. CdsRBD aligns the RNA to the RNA cleavage active site in the RNaseIII domain for efficient and high-fidelity siRNA production. This study reveals multi functions of Dicer-2 CdsRBD and sheds light on the molecular mechanism by which Dicer-2 produces 21 nt siRNAs with a high efficiency and fidelity for efficient RNA silencing.
Keywords
- Received December 1, 2016.
- Accepted April 10, 2017.
- Published by Cold Spring Harbor Laboratory Press for the RNA Society
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