<p> Dicer plays a central role in RNA interference pathways by cleaving double-stranded RNAs (dsRNAs) to produce small regulatory RNAs. Human Dicer can process long double-stranded and hairpin precursor RNAs to yield short interfering RNAs (siRNAs) or microRNAs (miRNAs), respectively. In humans, Argonaute2 (AGO2) assembles with the guide RNA-generating enzyme Dicer and either the RNA-binding protein TRBP or PACT to form a RISC-loading complex (RLC), which is necessary for efficient transfer of nascent siRNAs from Dicer to AGO2. Here, I have used electron microscopy and single particle analysis of human Dicer-RNA complexes and the RLC to gain insight into the structural basis for human Dicer's substrate preference and RISC-loading. My studies show that Dicer traps pre-siRNAs in a non-productive conformation, while interactions of Dicer with pre-miRNAs and dsRNA binding proteins induce structural changes in the enzyme that enable productive substrate recognition in the central catalytic channel. The RLC Dicer's N-terminal DExH/D domain, located in a short base branch, interacts with TRBP, whereas its C-terminal catalytic domains in the main body are proximal to AGO2. A model generated by docking the available atomic structures of Dicer and Argonaute homologs into the RLC reconstruction suggests a mechanism for siRNA transfer from Dicer to AGO2.</p>
| Identifer | oai:union.ndltd.org:PROQUEST/oai:pqdtoai.proquest.com:3578460 |
| Date | 26 February 2014 |
| Creators | Taylor, David W., Jr. |
| Publisher | Yale University |
| Source Sets | ProQuest.com |
| Language | English |
| Detected Language | English |
| Type | thesis |
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