Intrinsically Disordered dsRNA Binding Domain of Arabidopsis thaliana DCL1 Folds in the Presence of Substrate RNA

SUAREZ IP; HAILS G; GAUTO DF; BENOIT MPMH; BOISBOUVIER J; RASIA RM

Dublin

Congreso; Disordered Motifs and Domains in Cell Control; 2014

Biophysical Society

DCL1 is the ribonuclease that carries out miRNA biogenesis in plants. The enzyme has two tandem double stranded RNA binding domains (dsRBDs) in its C-terminus, which are essential for the enzyme function in vivo. By means of fluorescence anisotropy assays, we show that the first of these domains (DCL1-A) binds precursor RNA fragments when isolated, and cooperates with the second domain in the recognition of substrate RNA. Remarkably, despite showing RNA binding activity, DCL1-A is intrinsically disordered. We produced four different constructs of the protein, spanning the isolated domain and including surrounding regions. The domain is unstructured in every case. We explored different solution conditions to test what could lead the domain to acquire an ordered structure, and found that it folds when bound to its substrate dsRNA. By acquiring a set of standard NMR spectra, we assigned ca. 90% of the backbone resonances corresponding to the free unfolded and bound folded protein. Analysis of NMR data of the free protein shows it transiently explores secondary structure elements on the C-term end that could be essential for its capability of binding to the substrate. We have calculated the structure of the folded protein in complex with dsRNA employing CS-Rosetta. The structure corresponds to a canonical dsRBD, bearing some differences. One of the three RNA binding regions is missing, but affinity for the substrate is not affected. Finally in the presence of excess dsRNA we observe an intermediate unfolded bound species. ZZ exchange experiments show that this unfolded form is in slow exchange with the folded form. Based on these results, we propose a binding mechanism and discuss functional implications.