Dissecting the molecular principles of piRNA homeostasis

Small RNA-guided gene silencing pathways provide an important line of defense against foreign gene<U+FFFD>c elements like viruses and transposable elements. The PIWI-interac<U+FFFD>ng RNA (piRNA) pathway ensures that transposons are e<U+FB00>ec<U+FFFD>vely silenced at the transcrip<U+FFFD>onal and post-transcrip<U+FFFD>onal levels in animal gonads. The iden<U+FFFD>ty and quan<U+FFFD>ty of the 22-32nt piRNAs that serve as sequence-speci<U+FB01>c guides for Argonaute proteins of the PIWI class are cri<U+FFFD>cal for the piRNA pathway`s silencing speci<U+FB01>city and e<U+FB03>cacy. While the factors and molecular processes underlying the piRNA pathway have been iden<U+FFFD><U+FB01>ed, we lack a quan<U+FFFD>ta<U+FFFD>ve understanding of how these processes interact spa<U+FFFD>ally and temporally to de<U+FB01>ne the cellular piRNA pool. The goal of this project is to inves<U+FFFD>gate the cellular kine<U+FFFD>cs underlying piRNA homeostasis. To uncover the principles of piRNA homeostasis in living cells, we will adapt SLAMseq to cells with a func<U+FFFD>onal piRNA pathway. SLAMseq is a technology that combines metabolic labeling of cellular RNA molecules with nucleic acid chemistry and next- genera<U+FFFD>on sequencing. We will decipher the cellular rates, molecular features, and regula<U+FFFD>on of piRNA precursor transcrip<U+FFFD>on, piRNA processing, and piRNA turnover with unprecedented spa<U+FFFD>al and temporal resolu<U+FFFD>on using biochemical frac<U+FFFD>ona<U+FFFD>on and chemical gene<U+FFFD>c perturba<U+FFFD>on 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 The proposed research is based on the recently developed SLAMseq technology and an unpublished, immortalized cell line that we have derived from Drosophila germline stem cells (GSCs).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 gene<U+FFFD>c engineering, recapitulates all known aspects of germline piRNA biology. These include expression of three PIWI-clade proteins loaded with speci<U+FB01>c piRNA popula<U+FFFD>ons, ping-pong piRNA biogenesis, and Rhino-dependent piRNA cluster biology within heterochroma<U+FFFD>n. This will allow us to determine piRNA homeostasis parameters speci<U+FB01>c to di<U+FB00>erent PIWI-clade proteins and compare them to microRNA and siRNA homeostasis in the same cells for the <U+FB01>rst <U+FFFD>me. Using the derived piRNA homeostasis parameters, we will explore the molecular basis of the adap<U+FFFD>ve nature of the piRNA pathway and will explore whether a target-dependent piRNA degrada<U+FFFD>on system similar to the microRNA pathway exists. The proposed project is a collabora<U+FFFD>on 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 direc<U+FFFD>on at the intersec<U+FFFD>on of nucleic acid chemistry, RNA metabolism, and germline biology to elucidate the molecular basis for the e<U+FB00>ec<U+FFFD>ve repression of transposons; and we will broaden our understanding on the kine<U+FFFD>c principles of gene expression and heterochroma<U+FFFD>n transcrip<U+FFFD>on.

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