Structural and Functional Analysis of Human Dicer

MicroRNA, which are a class of single stranded non-coding RNA, play an important role in regulating gene expression and protein translation by targeting messenger RNA via the RNA Induced Silencing Complex (RISC). Beyond microRNA’s (miRNA or miR) general regulatory functions, these biological macromolecules also participate in preventing or promoting the development of disease phenotypes, most notably, human cancers. Acting as either tumor suppressors or oncogenic miRNAs (oncomiRs), miRNAs are capable of inhibiting or inducing cell apoptosis, proliferation, and/or metastasis among other cellular effects. With their clear connection to human health and disease onset, developing therapeutic methods to treat microRNA-related diseases has become a high priority. However, challenges in targeting microRNAs directly due to their flexible structure, lack of selectivity, and highly electronegative surface has hindered such efforts. In response to these challenges, taking a closer look at the proteins involved in miRNA maturation may provide a more feasible therapeutic route for treating miRNA-related illnesses. Within the canonical miRNA biogenesis pathway, two RNase III enzymes, Drosha and Dicer, perform critical cleavage reactions leading to the production of a mature miRNA strand. The RNase III endoribonuclease Dicer performs the final cytoplasmic cleavage reaction in which a precursor miRNA (pre-miRNA) is processed, and two mature microRNA strands are produced. Despite Dicer’s importance in the miRNA pathway, it was not until the resolution revolution of cryogenic electron microscopy (cryo-EM) that we obtained our first insights into full-length Dicer structure. Using this structural technique, numerous structures of Drosophila Dicer-1 and Dicer-2 in a variety of conformational and functional states bound to dsRNA substrates have been determined ranging in resolutions from 7Å to 3Å. However, structures depicting human Dicer (hsDicer) have been slow to advance with current structural information only showing apo and pre-cleavage Dicer conformations. In response to this lack of structural knowledge, the goal of this thesis was to determine the structure of hsDicer bound to pre-miRNA in a cleavage competent state as well as characterize the effects and differences in activity in the presence of both pre-miR and non-pre-miR substrates. To answer these questions regarding the underling mechanism(s) of hsDicer which are described in Chapter 1, the work outlined in this thesis utilized a mixture of structural and biochemical techniques. In Chapter 2, cryo-EM enabled the structural characterization of a partially inactive hsDicer mutant (D1709A) in complex with pre-miR-21. While in Chapter 3, cellular and in vitro functional studies were used to identify difference in hsDicer activity in the presence of Platform-PAZ disease-associated mutations. Furthermore, as a more complete picture of hsDicer activity, a pre-miR substrate (pre-miR-21) as well as a non-pre-miR substrates (snord37) were used to probe the activity landscape of hsDicer function. Results from these structural and functional studies have revealed that not only is hsDicer dependent on large conformational changes in its helicase domain to initiate the cleavage reaction, but that the mechanism(s) by which cleavage initiation occurs appears to be substrate dependent. Overall, these studies highlight the versatility of Dicer activity, but due to a lack of resolution, fall short in providing new molecular models of hsDicer.PHDMedicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/175619/1/torrezrm_1.pd