The double stranded RNA binding domains of A. thaliana DCL1

BURDISSO P; BOLOGNA NG; PALATNIK JF; BOISBOUVIER J; RASIA RM

Salta

Congreso; 3rd Latin American Protein Society Meeting; 2010

Ribonuclease III like enzymes in the dicer family are involved in different paths related to RNA silencing in plants. A. thaliana features four related enzymes called dicer like ribonucleases (DCL1-4). These enzymes share a common domain strucuture, which is: DexH-box RNA-helicase-C, DUF283, PAZ, two ribonuclease-III motifs and at least one double-stranded-RNA-binding domain (dsRBD) at the C-terminus. DCL1 produces the two cuts necessary to precisely excise mature mi-RNA from its precursors, pri-miRNA. Recognition of the substrate is presumably carried out by the dsRBDs of the protein and the DUF283 domain, which was recently shown to present a dsRBD-like fold2. DCL1 protein differs from the canonical Dicer proteins by the presence of a second dsRNA-binding domain at the C-terminus (dsRBDII). With the aim of understanding RNA recognition by DCL1 we characterized its dsRBDII. We produced and expressed the construction spanning this domain and obtained 1H-15N NMR correlation spectra. The protein appears to represent an independent folding unit. Optimization of the purification protocol was carried out to achieve a stable sample for NMR studies. Analysis of the secondary chemical shifts of the backbone resonances shows that the secondary structure is consistent with a dsRBD fold. Further studies of the RNA binding capabilities of the domain are being pursued in order to shed light on the molecular recognition features that govern miRNA production by DCL1.