Dynamic Modeling of Dicer Function

About This Grant

Project Summary It has been well established that small non-coding RNAs, such as microRNA (miRNA) and small interfering RNA (siRNA), act as potent regulators in numerous biological processes. MiRNA, for instance, modulate gene expression by targeting mRNA based on sequence complementary via the RNA Induced Silencing Complex (RISC). This allows miRNA to regulate key cellular events including cell differentiation, proliferation, and apoptosis. Consequently, when expression levels of miRNAs are dysregulated or alterations to the miRNA sequence occur, individuals can become more susceptible to the development of a variety of disease types, such as cancer, immune-related and neurodegenerative diseases. In response, researchers have been using a combination of techniques to identify the mechanism(s) behind points of dysregulation to guide future RNA- targeted therapeutics. Towards this goal, extensive studies have been carried out to identify and characterize the proteins involved in the miRNA biogenesis pathway, which include the endoribonuclease III protein Dicer. Dicer performs the final cleavage reaction to remove the hairpin loop region of precursor miRNA (pre-miRNA) to produce mature miRNA strands. While in humans, Dicer’s role is centralized around miRNA maturation and regulation, exploration into Dicer proteins from other species has revealed a highly dynamic and multifunctional activity, which includes the cleavage of siRNAs, a key component in antiviral defense. Differences in substrate specify are seemingly dependent on the ATP hydrolysis activity of Dicer’s N-terminal helicase domain. To further investigate this functionality and determine its connection to substrate specificity, structural and biochemical studies have been implemented to uncover the mechanisms behind ATP-independent vs ATP-dependent helicase activity. However, these approaches have been unable to capture the conformational heterogeneity associated with this functionality. Therefore, this proposal puts forth a new approach that combines results from a series of molecular dynamics simulations used to investigate Dicer’s flexibility (Aim 1) with published experimental observations from biochemical, kinetic, and structural studies to create data-informed predictive simulations (Aim 2). These predictive simulations will be used to investigate the central hypothesis of this proposal which is that the overall flexibility of Dicer’s helicase domain is dependent on whether it can hydrolyze ATP, and that in the absence of this activity, the helicase domain is unable to maintain one stable structure and samples a variety of conformational states. Overall, with the extensive resources offered by the University of Utah and expert guidance provided to me by my mentors, I am confident that this proposal will not only generate the first comprehensive integrative dynamic model of Dicer function, but also introduce a novel workflow to explore hypotheses and predict related functional outcomes of other disease-associated RNA interactions.