IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
RNA induced folding of the first double stranded RNA binding domain from A. thaliana DCL1.
Autor/es:
BURDISSO, PAULA; SUAREZ, IRINA; BENOIT, MATTHIEU; BOISBOUVIER, JEROME; RASIA, RODOLFO M.
Lugar:
Rio de Janeiro
Reunión:
Congreso; 18 th ISMAR Meeting; 2013
Resumen:
MicroRNAs are essential gene regulators in multicellular organisms. Plant miRNAs are processed in the nucleus by a protein complex formed by DICER-LIKE1 (DCL1), HYL1 and SERRATE. DCL1 has a central role in the recognition and processing of the heterogeneous plant precursors. Little is known about the structural aspects of this protein. In a previous work, we characterized the second dsRBD of DCL1. Here we present a structural characterization of the first DCL1 double stranded RNA binding domain. In contrast with canonical dsRBDs, this domain binds dsDNA with a similar affinity as dsRNA, the same as DCL1-dsRBD2. We found that both domains cooperate for substrate binding, but not for DNA binding. Quite unexpectedly we found that dsRBD1 is intrinsically disordered, even in the context of flanking domains, but folds upon binding substrate RNA. We have assigned the backbone resonances corresponding to the free unfolded and bound folded protein. The free protein, though intrinsically disordered, shows a tendency to populate folded conformations that correspond to those expected for a dsRBD. The assignment of the bound form allowed us to calculate the fold of the protein in complex with dsRNA employing CS-Rosetta. This analysis shows that this domain acquires the dsRBD fold when bound to substrate. We further found that the negatively charged surface provided by SDS micelles shifts the protein towards folded conformations, but these are different from that found in the complex with RNA. Experimental comparison with dsRBDs from other Dicer proteins show that the intrinsically disorder nature is exclusive of DCL1-dsRBD1. Sequence analysis and in vivo function of the protein suggests that in this domain the instability of the free form and the ability to fold upon binding the substrate have to be essential for the function of the whole DCL1 enzyme in miRNA processing.