CHARACTERIZATION OF THE RNA BINDING DOMAINS OF ARABIDOPSIS THALIANA DCL1

BURDISSO, PAULA; BOLOGNA, NICOLAS G.; PALATNIK, JAVIER F.; BOISBOUVIER, JEROME; RASIA, RODOLFO M.

Buzios

Congreso; VII Congreso Iberoamericano de Biofìsica; 2009

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: N-terminal DexH-box  RNA-helicase-C motifs, a DUF283 domain (domain of unknown function),  a PAZ (Piwi/Argonaute/Zwille) domain, two ribonuclease-III motifs and at  least one double-stranded-RNA (dsRNA) binding domain. DCL1 produces  the cuts necessary to precisely excise mature mi-RNA from its precursors.  Recognition of the substrate is presumably carried out by  dsRNA binding domains and DUF283 domain, which was recently  classified as an RNA binding domain based on remote sequence  homology. DCL1 protein differs from the canonical Dicer proteins by the  presence of a second dsRNA- binding domain. With the aim of  understanding RNA recognition by DCL1 we characterized the annotated RNA binding domains. We produced and expressed three different  constructions spanning the individual C-terminal dsRBDs and the double  domain and obtained 1H-15N NMR correlation spectra of the  constructions. Whereas the second dsRBD appears to represent an  independent folding unit, the first dsRBD does not give a folded  polypeptide, even in context of the double domain construct, suggesting  that interaction of the first domain with the preceding RNAse III domain  is necessary for it to fold. We investigated a DUF283 construct as well,  which gives also a folded protein. Analysis of the secondary chemical shifts of the backbone resonances of DUF 283 shows that this domain has secondary structure consistent with a dsRBD fold. Further studies of the RNA binding capabilities of the domains are being pursued in order to shed light on the molecular recognition features that govern miRNA production by DCL1.