PROJECT SUMMARY In the past twenty years, thousands of non-coding RNAs (ncRNAs) have been discovered as potential regulators of gene expression. Within this group, microRNAs (miRNAs) have emerged as essential mediators of post-transcriptional gene regulation, and defects in specific miRNA pathways have been linked to numerous human diseases. While a basic understanding of how miRNAs are expressed and function has been achieved, outstanding questions regarding the regulation of miRNA biogenesis and target recognition in vivo remain to be solved. Caenorhabditis elegans worms have proven to be an advantageous model to investigate miRNA biology at the organismal level. The development of sensitive biochemical methods, unique worm strains, extensive genomic datasets, and robust computational pipelines has enabled novel insights into miRNA expression and targeting in the context of a developing animal. Utilizing and further innovating these resources and methods, the proposed research will contribute to a better understanding of how miRNAs find and regulate targets within a live animal under ideal as well as stressful conditions. With a confident set of targets bound by the miRNA complex in vivo, features that underlie the different mechanisms deployed to regulate them will be deduced. The identification of numerous ncRNAs, including miRNAs and long non-coding RNAs (lncRNAs), induced by heat shock in C. elegans sets a foundation for discovering new mechanisms for regulating gene expression in response to this stress. The discovery that the miRNA pathway plays a key role during heat shock recovery, highlights the importance of this under-explored phase of the heat shock response. The current research is focused on elucidating how the expression of specific miRNAs and lncRNAs is regulated by heat shock and, in turn, how these ncRNAs function to protect the organism during this stress. Over the next five years, these studies have the potential to reveal novel roles for ncRNAs in response to heat shock and set the stage for investigating the impact of ncRNA pathways in the organismal response to other stresses, including disease states. The long-term goal of this research program is to contribute new insights into how ncRNAs control gene expression under varied conditions in an intact organism. Furthermore, knowledge gained from these studies has the potential for significant impact on the design and utilization of RNA-based therapeutics for the treatment of human disease.