Dissecting the molecular principles of piRNA homeostasis

Small RNA-guided gene silencing pathways provide an important line of defense against foreign genec elements like viruses and transposable elements. The PIWI-interacng RNA (piRNA) pathway ensures that transposons are eecvely silenced at the transcriponal and post-transcriponal levels in animal gonads. The identy and quanty of the 22-32nt piRNAs that serve as sequence-specic guides for Argonaute proteins of the PIWI class are crical for the piRNA pathway`s silencing specicity and ecacy. While the factors and molecular processes underlying the piRNA pathway have been idened, we lack a quantave understanding of how these processes interact spaally and temporally to dene the cellular piRNA pool. The goal of this project is to invesgate the cellular kinecs underlying piRNA homeostasis. To uncover the principles of piRNA homeostasis in living cells, we will adapt SLAMseq to cells with a funconal piRNA pathway. SLAMseq is a technology that combines metabolic labeling of cellular RNA molecules with nucleic acid chemistry and next- generaon sequencing. We will decipher the cellular rates, molecular features, and regulaon of piRNA precursor transcripon, piRNA processing, and piRNA turnover with unprecedented spaal and temporal resoluon using biochemical fraconaon and chemical genec perturbaon of key piRNA pathway factors. The proposed research is based on the recently developed SLAMseq technology and an unpublished, immortalized cell line that we have derived from Drosophila germline stem cells (GSCs). The GSC line, in which we established state-of-the-art genec engineering, recapitulates all known aspects of germline piRNA biology. These include expression of three PIWI-clade proteins loaded with specic piRNA populaons, ping-pong piRNA biogenesis, and Rhino-dependent piRNA cluster biology within heterochroman. This will allow us to determine piRNA homeostasis parameters specic to dierent PIWI-clade proteins and compare them to microRNA and siRNA homeostasis in the same cells for the rst me. Using the derived piRNA homeostasis parameters, we will explore the molecular basis of the adapve nature of the piRNA pathway and will explore whether a target-dependent piRNA degradaon system similar to the microRNA pathway exists. The proposed project is a collaboraon between the groups of J. Brennecke and S. Ameres and will leverage the complementary technical and conceptual experience of both groups. We will pioneer a novel research direcon at the intersecon of nucleic acid chemistry, RNA metabolism, and germline biology to elucidate the molecular basis for the eecve repression of transposons; and we will broaden our understanding on the kinec principles of gene expression and heterochroman transcripon.