THE DOUBLE STRANDED RNA BINDING DOMAINS FROM Arabnidosis thaliana DCL1

SUAREZ IP; BURDISSO P; RASIA RM

Congreso; XLVIII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2012

Biogenesis of small RNAs is a complex process involving ribonuclease III like enzymes of the dicer family. 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 precursors, pri-miRNA. 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). The construction expressing DCL1-dsRBD2 gives a well folded protein. We obtained the structure of this domain using solution NMR. DCL1-dsRBD2 shows some significant differences when compared to canonical dsRBDs: an insertion in loop beta2-beta3 that could be involved in protein-protein interaction, and a shorter helix 1 which could give rise to higher versatility in substrate specificity. For DCL1-dsRBD1 we produced four constructs spanning the annotated domain alone and including surrounding regions. The domain is intrinsically disordered in every case. We explored different conditions and found evidence that it folds in the presence of RNA. Analysis of NMR data of the free protein shows it has no tendency to acquire secondary structure. We have calculated the structure of the protein in complex with dsRNA employing CS- Rosetta.