Characterization of PAZ/PIWI domain proteins by identification of their binding partners

RNA interference (RNAi) is the regulation of gene expression mediated by double-stranded RNA. Cells silence genes by RNAi either transcriptionally or post-transcriptionally through mRNA degradation or translational inhibition. My thesis work has focused on characterization of the PAZ/PIWI Domain-containing (PPD) proteins via identification of their binding partners. PPD proteins play key roles in RNAi and function during both initiation and effector steps. The amino terminal region of rat Ago2 (rAgo2), a PPD protein, interacts with an Hsp90 heterocomplex, a process that is required for the stability of rAgo2 and its translocation to membranes. Next, the interaction between the human orthologue of rAgo2 (hAgo2) and a distantly related PPD family member (Hiwi) with the RNase III enzyme Dicer was explored. The results indicate that the PIWI domains of PPD proteins interact with the RNase III domain of Dicer. Mapping of the interaction sites suggested that the strongest interaction occurs between a highly conserved 43 amino acid long sequence called the PIWI-box and the RNase III-A domain of Dicer. Furthermore, the interactions between PPD proteins and Dicer are direct, and require Hsp90 activity. The interaction of PPD proteins with Dicer down-regulates the activity of this enzyme. I have developed a model in which stable Dicer-PPD complexes function at the initiation and effector steps of RNAi.