PARENT PROJECT SUMMARY Despite their diminutive length, 20-30-nucleotide-long small RNAs affect nearly all developmental and disease processes, from fertility in flies to cancer in humans. Small RNA associate with Argonaute proteins to direct sequence-specific degradation or translational repression of matching mRNAs in a process called RNA interference (RNAi). Small RNAs can also function in an alternative mode to promote gene expression. Within the germline of the tiny nematode worm, Caenorhabditis elegans, two broad classes of small RNAs – piwi- interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) – interact with nearly all genes, silencing some and promoting the expression of others. Remarkably, some piRNAs and siRNAs are transmitted from one generation to the next, providing a heritable mechanism for regulating gene expression without changes to the underlying DNA. In C. elegans, piRNAs and siRNAs are required for optimal fertility and germline immortality. We and others identified a role for maternally-derived piRNAs and siRNAs in protecting essential genes from silencing. We also showed that these small RNAs have a role in establishing proper gene expression in the embryo that is crucial throughout development, although the mechanism underlying this phenomenon requires further study. To identify the roles of piRNAs and siRNAs in ensuring proper gene expression from one generation to the next, we will address two related questions: 1) How do piRNAs and siRNAs regulate gene expression to promote fertility and germline immortality? and 2) What are the molecular roles of maternally deposited piRNAs and siRNAs? A second area of my research centers on gene regulatory mechanisms involving a third class of small RNAs, called microRNAs (miRNAs). We recently uncovered a distinct branch of the miRNA pathway required for proper developmental timing and optimal fertility in the germline. How this pathway regulates gene expression is an important area of future research. This relates to the third question we will address: 3) How do miRNAs regulate developmental timing in the germline? miRNAs are processed from a limited number of transcripts that form hairpin-like secondary structures. The hairpin itself is not sufficient to mark a transcript for processing, and in many species, including humans, primary transcripts contain additional sequence elements that promote miRNA formation. Not all miRNA transcripts contain these elements and in some animals, including C. elegans, they are completely lacking. We developed a sensor that reports on miRNA transcript recognition. Using the sensor, we will address a fourth question: 4) How are miRNA transcripts distinguished from other RNAs in C. elegans? The ease in which genetics, genome editing, and genomics assays can be done in C. elegans makes it an ideal system to address these four questions. The mechanism of miRNA formation and the molecular roles of miRNAs, piRNAs, and siRNAs are highly conserved i...