Understanding what regulates a regulator: the molecular basis for homeostatic small RNA-mediated gene regulation

PROJECT SUMMARY/ABSTRACT RNA interference (RNAi) pathways are critical regulators of cellular programs conserved across eukaryotes. Target-specific RNAi-mediated regulation for thousands of loci is achieved with a small set of Argonaute (AGO) proteins, each complexed with many diverse small RNAs. RNAi defects trigger catastrophic mis-regulation of gene expression associated with genetic diseases, tumorigenesis, aberrant development, and sterility. Moreover, disruption to RNAi-mediated gene regulation impacts maintenance of cellular identity and allows de- repressed transposons to wreak havoc on the genome. Thus, a mechanistic understanding of the highly complex molecular mechanisms of RNAi pathways, and how they are impacted by real-world stresses, is fundamental to advancing our knowledge of small RNAs in human health and disease. While we understand the basic mechanisms of RNAi-mediated regulation we know very little about how RNAi pathways are controlled to preserve appropriate gene expression. After all, what regulates a regulatory network? Mechanisms of RNAi homeostasis have remained largely unexplored, in part because studying RNAi homeostasis without functionally inactivating one or more RNAi factor has not been feasible. Our preliminary work identified the first two feedback mechanisms that maintain balance amongst the RNAi pathway branches. One of these feedback motifs enabled us to create a novel mutant that makes studying the effects of disrupting RNAi homeostasis without altering the function of any factor possible. Experiments leveraging this mutant revealed that perturbations to RNAi homeostasis negatively impacts a wide range of physiological processes. In this proposal, we build upon these discoveries to address three fundamental questions with the goal of advancing our understanding of how RNAi-mediated feedback regulates RNAi function to maintain cellular homeostasis. The questions will explore the mechanisms of target transcript sorting amongst distinct RNAi branches, how RNAi pathways are temporally regulated throughout development, and the molecular and physiological consequences of disrupting RNAi homeostasis. Findings from this work will enable us to build a comprehensive model of the feedback regulatory architecture that coordinates the overlapping network of RNAi pathways. Additionally, this work will further our understanding of how real-world stressors impact gene regulation. Ultimately, this work will transform our understanding of RNAi by establishing mechanistic knowledge of how RNAi homeostasis facilities the interactions between RNAi and other cellular pathways that coordinate many physiological processes. Addressing these fundamental questions is critical for our understanding of small RNAs in human health and is important for the development of bioengineering techniques that harness the power of our own regulatory networks for use in therapeutic synthetic gene regulation. Therefore, findings from the proposed work will support the National Institute of Health mission, which aims to foster fundamental discoveries that aid in protecting and improving human health.