RNA interference (RNAi) pathways, consisting of Argonaute proteins and small RNAs that provide sequence specificity, play key roles in gene regulation across all domains of life. Studies of Argonautes in many species have revealed that they impact gene expression at nearly every stage in the life cycle of a transcript—from transcription to translation. Due to their central role in RNAi and profound impact on development and differentiation in numerous organisms, uncovering new and conserved molecular mechanisms of Argonautes advances our fundamental knowledge of cellular function and has the potential to provide more precise means to manipulate gene expression, relevant for biotechnology and therapeutics.The C. elegans genome encodes an expanded family of 27 ago genes, 19 of which produce functional proteins. In this thesis I have undertaken a systematic study of every functional C. elegans Argonaute to develop a comprehensive portrait of the molecular mechanisms of these mostly uncharacterized proteins throughout development. Using CRISPR/Cas9 genome-editing, I epitope-tagged each Argonaute with GFP::3xFLAG. I used confocal microscopy to characterize the expression patterns of each Argonaute throughout development and defined the tissue specific and subcellular localizations of each Argonaute. High-throughput sequencing of 1) small RNAs associated with each Argonaute, and 2) total small RNA pools from argonaute mutants versus wild type uncovered the stratification of subsets of Argonautes into distinct gene regulatory modules. Directed by the localization and small RNA sequencing data, I employed phenotypic analyses of argonaute mutants under normal and stressful conditions. These revealed previously unappreciated phenotypes, including a transgenerational loss of fertility known as the Mortal Germline (mrt) defect, and resistance to the bacterial pathogen Pseudomonas aeruginosa. Overall, my systematic and pioneering studies provide an unprecedented view of the small RNA regulatory landscape throughout the development of a complex animal.Ph.D
