The role of Loquacious-PD in small RNA biogenesis in Drosophila melanogaster

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Title The role of Loquacious-PD in small RNA biogenesis in Drosophila melanogaster
Publication Type dissertation
School or College School of Medicine
Department Biochemistry
Author Trettin, Kyle Daniel
Date 2018
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.
Type Text
Publisher University of Utah
Subject Biochemistry
Dissertation Name Doctor of Philosophy
Language eng
Rights Management (c) Kyle Daniel Trettin
Format application/pdf
Format Medium application/pdf
ARK ark:/87278/s6rv5qw6
Setname ir_etd
ID 1526104
Reference URL https://collections.lib.utah.edu/ark:/87278/s6rv5qw6
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