| Περίληψη: | From prokaryotic CRISPR to eukaryotic RNA interference (RNAi), small RNA-based mechanisms have been universally employed to defend the host against genome invaders, including viruses and transposable elements. Transposons and endogenous retroviruses are genomic DNA sequences that can change their position within the genome (transposition), causing mutagenesis and threatening genome stability. In animal gonads, a germline-specific class of small RNAs, associate with the PIWI subfamily of Argonaute proteins (PIWI-interacting RNAs, piRNAs) and guide the suppression of transposable elements on both transcriptional and post-transcriptional level. From invertebrates to mammals, the piRNA pathway is essential for germ cell development and fertility. During piRNA biogenesis, large genomic loci (piRNA clusters) produce long single stranded RNA precursors which are processed into thousands of unique piRNA sequences in a non-predictable manner. As a result, millions of different piRNAs have been observed in flies and mice. This remarkable diversity combined with the tolerated mismatches during target-recognition, pose a challenge to understand the non-self specificity of the piRNA-mediated genome defense. Here we investigated mechanisms of piRNA-guided transcriptional silencing through a combination of molecular, genetic, and computational approaches. We particularly aimed to understand how the diverse population of piRNAs avoids off-target events that would permanently shut down essential host genes. To accomplish this goal, we developed a quantitative piRNA-sequencing method and a direct reporter assay to measure piRNA-guided silencing at single cell level. Our work revealed that universally the abundance of unique piRNA sequences is highly skewed and only a few of the most-abundant piRNAs are present in every cell. We observe that individual piRNA abundance is regulated during the biogenesis step and is determined by the sequence preferences of the Zucchini processor. Our direct reporter assay demonstrated that efficient silencing of a target correlates with the combined abundance of antisense piRNAs and allowed us to observe cell to cell differences in piRNA-mediated transcriptional silencing. The vast majority of the diverse piRNA sequences occur sporadically and establish cell to cell diversity. Biogenesis of these sporadic piRNAs could be indicative of a mechanism that allows adaptation to new invaders. Furthermore, by establishing cell to cell diversity, piRNAs could be promoting reproductive polymorphism and increase the chances of species survival under evolutionary pressure. A direct consequence of our study is that piRNA abundance becomes a key consideration when studying mechanisms of piRNA function, biogenesis and targeting. Our results also emphasized the importance of high-quality, quantitative piRNA datasets that accurately distinguish between rare and abundant piRNA sequences. Thus, during this PhD, we also established a simple and reliable method for the preparation of high-quality piRNA sequencing libraries. Our method has eliminated the need for radioactive labeling and UREA-polyacrylamide gel purification, and provided an unbiased view of the complete bona fide piRNA population in Drosophila. With our improved method, we characterized Piwi-piRNA complexes in the different cell lineages of the Drosophila ovary. We observe that the same PIWI protein targets different transposons in the germ cells and in the follicle cells of the ovary. Upon association with a piRNA, this PIWI protein enters the nucleus and is responsible for the de novo epigenetic silencing of active transposons. Follicle PIWI-piRNA complexes target almost exclusively a single family of retrotransposons, namely Gypsy, while the corresponding germ cell complexes target a broader range of retrotransposon families and different members of the Gypsy family. This observation is linked to the differential expression of piRNA-generating clusters in the two ovarian cell types. How this differential expression of piRNA-generating regions is initially established and how are the transposons that are neglected by the piRNA pathway in the different compartments controlled, are open questions that would require further investigation. In summary my PhD work provides novel insight into key questions regarding the piRNA-mediated transcriptional silencing and it created resources and foundations for future piRNA studies.
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