Investigating the role of Piwi in Drosophila germline specification - PROJECT SUMMARY Evolutionary success relies on an animal’s ability to produce reproductive cells, called germ cells, that can generate an entirely new organism. Across diverse species, germ cell fate and function are controlled by the PIWI/piRNA pathway. PIWI proteins are RNA-binding proteins that partner with small noncoding PIWI-interacting RNAs (piRNAs) to regulate their RNA targets. Although their best characterized function is transposon silencing, emerging evidence posits that PIWI proteins can directly regulate mRNAs. Importantly, in humans PIWI proteins are normally enriched in germ cells but are aberrantly found in many cancers. Therefore, elucidating how PIWI protein regulate the genome is critical for understanding dysregulation of gene expression in human disease. In addition to use of the PIWI/piRNA pathway, germ cells across species exhibit common cytoplasmic hallmarks: RNA-protein condensates called germ granules. Across diverse species, germ granules are hubs for post- transcriptional regulation throughout the germ cell life cycle and contain conserved germ cell proteins, including PIWI proteins. As germ granules are critical for germ cell function, they have recently emerged as an informative paradigm for the cellular consequences of post-transcriptional regulation in RNA granules. This proposal focuses on early Drosophila embryos, which contain germ granules, called polar granules, at the posterior pole. Polar granules are assemblies of maternally loaded proteins and RNAs, including PIWI proteins, that control formation of embryonic primordial germ cells (PGCs), the earliest precursors of the germline. Although is it known that Piwi, a specific protein in the Drosophila PIWI family, is crucial for PGC formation, the mechanisms underlying its function in germline specification are only partially understood. I hypothesize that Piwi controls PGC formation by regulating the stability and translation of key mRNAs within polar granules. In this proposed project, I will investigate the role of Piwi polar granule assembly and post-transcriptional regulation of polar granule mRNAs. First, I will use Structured Illumination Microscopy (SIM) to analyze Piwi localization within polar granules and the consequences of Piwi depletion on polar granule architecture. A similar approach in a transgenic form of Piwi that cannot bind piRNAs will determine whether Piwi functions in germline specification via piRNAs. Next, I will perform individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) to identify Piwi-target mRNAs in embryos. Downstream analysis using single-molecule fluorescence in situ hybridization (smFISH) and the Suntag system to fluorescently visualize nascent peptides will determine whether Piwi-target mRNAs exhibit altered localization, stability, or translation in the absence of Piwi or when Piwi cannot bind piRNAs. The proposed work will elucidate how Piwi, which is best characterized in adult germ cells, controls the initial formation of the germline during embryogenesis. More broadly, I will investigate how Piwi uses piRNAs to directly regulate mRNAs within cytoplasmic RNA granules, with potential broad implications for human diseases in which PIWI proteins are aberrantly expressed.
