CONICET | Buscador de Institutos y Recursos Humanos

Biogenesis of small RNAs is a complex process involving ribonuclease III like enzymes of the dicer family1. In A. thaliana the processing of miRNA is carried out exclusively by DCL1, which produces the two cuts necessary to precisely excise mature miRNA from its longer precursors, pri-miRNA2. Structural features which allow DCL1 to process this heterogeneous group of precursors still remain to be elucidated. In order to understand RNA recognition by DCL1 we studied both of its double-stranded RNA binding domains (dsRBDs) from a structural perspective. Both domains are located in tandem in the C-terminal region; the second dsRBD is not present in other dicer proteins3. The construct expressing DCL1-dsRBD2 gives a well folded protein. We obtained the structure of this domain employing solution NMR. DCL1-dsRBD2 shows some significant differences when compared to canonical dsRBDs. For DCL1-dsRBD1 we produced four constructs spanning the annotated domain alone and including surrounding regions. The domain is unstructured in every case. We explored different solution conditions and additives to test what can lead the domain to acquire an ordered structure, and found that it folds only in the presence of substrate RNA. We have assigned the backbone resonances corresponding to the free unfolded and bound folded protein. Analysis of NMR data of the free protein shows it explores helical conformations in the region corresponding to helix 2. The assignment of the bound form allowed us to calculate the fold of the protein in complex with dsRNA employing CS-Rosetta. Both domains bind dsDNA as well as dsRNA with similar affinities, an unusual feature for dsRBDs. However, the double domain has a higher selectivity for dsRNA, suggesting that both domains cooperate in substrate recognition. Altogether our results show that the function of this region of DCL1 is more complex than anticipated by sequence annotation.