Project Summary RNA interference (RNAi) has driven a surge of research over the past twenty years as a powerful tool for gene silencing, and the discovery of a distinct class of noncoding RNAs, microRNAs (miRNAs), has revolutionized our outlook of genome regulation. miRNAs bind to a critical effector molecule, the Argonaute protein, to achieve sequence-specific recognition and repression of their target. Because the rules for complementary recognition seem simple and predictable, the dysregulation of miRNAs has been implicated as the sole culprit of disease in over 40,000 articles on PubMed. The sheer volume of publications suggest that miRNAs have omnipotent control over gene expression. This model of ubiquitous miRNA regulation suggested that there is an urgent need for a fundamental reexamination of the available gene silencing infrastructure present in a cell. Progress in identifying high-quality candidates for miRNA-based therapies have been hindered by this major barrier that miRNAs are expected to recognize more targets in a cell than there are catalytic molecules to guide. This project seeks to answer the question of how the competition for shared gene silencing resources can have a dramatic impact on the number of targets miRNAs can control. The first and second aim will both use quantitative mass spectrometry (MS) to count the AGO molecules per cell and RNA-sequencing to determine the miRNA and mRNA expression in a HCT116 colon cancer model system. While the first aim will interrogate CRISPR-mediated AGO knockout cell lines, the second aim will provide the complementary overexpression analysis. Furthermore, these cell lines will be used as rigorous controls for unbiased identification of the AGOs’ respective RNA binding sites using AGO-eCLIP-Seq. The third aim is to use three types of cell stress, nutrient deprivation, oxidative stress, and hypoxia, to trigger an endogenous RNAi response, which will be assessed by AGO copy number with quantitative MS, miRNA and target expression with RNA-Seq techniques, and RNA binding locations with AGO-eCLIP-Seq. The multi-disciplinary environment in the Corey laboratory fosters collaboration amongst diverse backgrounds, and trainees benefit from Dr. Corey’s attentive mentorship and the guidance of several established research scientists. By the end of this training fellowship period, I will accomplish four goals: 1) to be knowledgeable about the literature in my chosen field of RNA biology, 2) to be comfortable writing grant proposals and manuscripts, 3) to be technically competent, and 4) speak with clarity to all audiences.

Project Narrative miRNAs have been implicated as the sole culprit influencing nearly every mRNA in the cell, yet early results suggest that RNA interference (RNAi) may have a more modest effect than dogma would suggest. This project aims to use a quantitative analysis of the core effector molecule of RNAi, the Argonaute proteins, to provide a fundamental reexamination of old assumptions about the abundance of effector, guide, and target molecules present in a cell. A deeper understanding of the gene silencing infrastructure available for targeting by miRNAs will facilitate progress towards defining the boundaries of miRNA control and eventually the development of miRNA-based therapies.