Project summary Organism development and cellular homeostasis depend on tightly regulated gene expression programs. Disruptions in gene regulation lead to developmental defects and contribute to human diseases including cancer and neurodegenerative disorders. MicroRNAs (miRNAs), small non-coding RNAs, play a critical role in post-transcriptional regulation of gene expression by repressing target genes. Dysregulation of miRNA activity can lead to disproportionate gene silencing or overexpression, with damaging consequences to organism development and health. Despite the significant role of miRNA function in development and disease, our understanding of how miRNA activity is regulated to maintain appropriate levels of gene repression remains incomplete. The overarching goal of this research is to understand how the gene-regulatory activity of miRNAs is controlled within the complex context of a developing organism. More specifically, we will investigate how miRNA target specificity is established through regulated, miRNA strand specific loading of the miRNA induced silencing complex (miRISC) and how key miRISC components and cofactors impact gene-regulatory miRNA activity. We will continue to leverage the power of C. elegans and will, through a range of methodologies, delineate the in vivo genetic and molecular networks regulating miRNA activity at steps of miRNA biogenesis and target repression. This research is expected to lay a foundation for understanding how gene regulatory programs are maintained during organism development through robust regulation of miRNA activity and how dysregulation of miRNA function leads to loss of gene expression control in disease.