IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
The dsRNA binding domains from A. thaliana DCL1
Autor/es:
SUAREZ, IRINA; BURDISSO, PAULA; PALATNIK, JAVIER F.; RASIA, RODOLFO M.
Lugar:
Alta Gracia
Reunión:
Workshop; Workshop Magnetic Resonance in a Cordubensis Perspective: New developments in NMR.; 2011
Resumen:
<!-- @page { margin: 0.79in } P { margin-bottom: 0.08in } --> <!-- @page { margin: 0.79in } H1 { margin-bottom: 0.04in; text-align: center } H1.western { font-family: "Arial", sans-serif; font-size: 12pt; so-language: en-US } H1.cjk { font-family: "DejaVu Sans"; font-size: 12pt } H1.ctl { font-family: "Arial", sans-serif; font-size: 12pt; font-weight: normal } P { margin-bottom: 0.08in } --> The DSRNA binding domains from A. thaliana DCL1 I.P. Suarez*, P. Burdisso, J.F. Palatnik, R.M. Rasia Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET) y Facultad de Ciencias Bioquímicas y Farmacéuticas UNR, Rosario, Argentina e-mail:isuarez@ibr.gov.ar The biogenesis of small RNAs is a complex process involving ribonuclease III like enzymes of the dicer family[1]. In A. thaliana four such enzymes, called DCL1-4 participate in different paths leading to the production of the different families of small RNAs (miRNAs, siRNAs, piRNAs)[2]. 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[3]. Recognition of the substrate is carried out by the RNA binding domains of the protein. DCL1 protein differs from the canonical Dicer proteins by the presence of a second dsRNA-binding domain at the C-terminus (DCL1-dsRBD2)[2]. In order to understand RNA recognition by DCL1 we studied the two dsRNA binding domains of DCL1. Both domains are located in tandem in the C-terminal region of the protein. We first produced several constructions spanning different parts of DCL1 C-terminus. The construction expressing DCL1-dsRBD2 gives a well folded protein. We assigned the HN, Ha, N, C´, CA and CB protein backbone resonances and acquired Residual Dipolar Coupling (RDC) data on C12E5/hexanol anisotropic phase. The orientational restraints were used to calculate the fold of the protein, which shows some significant differences when compared to canonical dsRBDs: an insertion in loop beta2-beta3 and a change in the orientation of helix 1. This results in a substantial displacement of the relative position of the putative RNA-binding sites, which could give rise to an atypical substrate specificity for this domain. For DCL1-dsRBD1 we produced four constructs including the annotated domain alone and the domain with sourrounding regions. Analysis of the 1H-15N HSQC spectra of the constructs show that the domain is intrinsically disordered in all of them. We explored different solution conditions and additives to test what can lead the domain to acquire an ordered structure, and found evidence that it folds in the presence of RNA. REFERENCES: Rana TM. Nat Rev Mol Cell Biol 8, 23-36 (2007) Margis R, Fusaro AS, Smith NA, Curtin SJ, Watson JM, Finnegan EJ, Waterhouse PM. FEBS Letters 580, 2442-2450 (2006) Xie Z, Khanna K, and Ruan S. Semin Cell Dev Biol 21, 790-797 (2010)