| Description |
RNA interference (RNAi) is an antiviral defense mechanism in many invertebrates. In Drosophila melanogaster, viral double-stranded RNA (dsRNA) is cleaved by Dicer-2 (Dcr-2) to produce antiviral short interfering RNAs (siRNAs). Dcr-2-dependent siRNAs are also produced from endogenous dsRNA but require an additional dsRNA-binding protein, Loquacious-PD (Loqs-PD). Previous biochemical characterization of termini-dependent cleavage of dsRNA by Dcr-2 suggests endo-siRNA precursors may be poor substrates given their varied terminal features. Prior to the work presented in this dissertation, it was unclear how Loqs-PD facilitates Dcr-2 cleavage of endo-siRNA precursors. The studies presented here provide mechanistic insight into the function of Loqs-PD during endo-siRNA biogenesis. In Chapter 2, the effect of Loqs-PD on termini-dependent cleavage by Dcr-2 is investigated. The cleavage rate of dsRNAs with either blunt (BLT) or 2-nucleotide 3' overhangs (3'ovr) by Dcr-2 is dramatically enhanced in the presence of Loqs-PD. Furthermore, the difference in cleavage rates between BLT and 3'ovr dsRNA is reduced, suggesting Loqs-PD reduces the termini-dependence of Dcr-2. Substrates containing terminal features predicted to occur in endo-siRNA precursors are found to require Loqs-PD for cleavage by Dcr-2. Finally, the effects of Loqs-PD are ATP-dependent, suggesting Loqs-PD elicits its function through Dcr-2's helicase domain. The studies presented in Chapter 2 indicate Loqs-PD directly modifies the termini-dependence of Dcr-2 to enable cleavage of suboptimal substrates. In Chapter 3, the mechanism of Loqs-PD-dependent siRNA biogenesis is explored. The ability to enhance cleavage of optimal and suboptimal substrates, bind dsRNA, and interact with Dcr-2 is tested for Loqs-PD and a series of truncations. The C-terminus of Loqs-PD mediates the interaction with Dcr-2 and is required to enhance cleavage of all substrates. The Loqs-PD-binding site on Dcr-2 is mapped to the Hel2 helicase subdomain. A dsRNA-binding mutant of Loqs-PD is unable to enhance Dcr-2 activity, despite retaining the ability to interact with Dcr-2. Building on the studies described in Chapter 2, a mechanistic model details how Loqs-PD couples dsRNA-binding and association with Dcr-2's helicase to promote cleavage of suboptimal substrates in a termini-independent manner. Finally, Chapter 4 explores questions posed by the studies presented in this dissertation and provides suggestions for future experiments. |
