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Mechanisms governing transposable element expression in the Drosophila germ line https://doi.org/10.17863/CAM.116718 Blake, Eleanor Karam Teixeira, Felipe Transposable element activity represents a critical threat to host organisms due to its potential to damage the genome and disrupt gene expression. Transposons are known to be particularly active in the developmental context of the germ line, to fulfil their selfish drive to increase in copy number in the genome that will be passed to the next generation. Due to this threat, dedicated mechanisms have evolved to control the expression of transposons in the germ line, notably including the PIWI-interacting RNA (piRNA) pathway, which acts to repress its targets through both chromatin-mediated silencing and post-transcriptional slicing. The P-element is a classic example of an active transposon that has been highly successful in invading new populations and subsequently increasing its copy number in Drosophila. The P-element was first identified through the severe impact of its activity on the development of germ cells, highlighting the need for host cell mechanisms to suppress transposon expression. Previous work has shown key differences between the regulation of the P-element and other transposons by the piRNA pathway. Notably, the dominant mechanism suppressing Pelement activity in germ cells is the regulation of its splicing through a currently unknown mechanism. This splicing regulation prevents the expression of the P-transposase enzyme, which catalyses the transposition of this transposon family. Furthermore, P-element splicing regulation depends on components of the piRNA pathway that are known to induce transcriptional silencing, but mysteriously, the levels of P-element transcripts seem to be barely decreased in the presence of piRNA targeting. Therefore, much remains unknown about the mechanisms underpinning P-element regulation by the piRNA pathway. In this thesis, I aim to further dissect the regulation of the P-element by the piRNA pathway as a means to better understand the regulatory mechanisms protecting the genome against transposons. To do this, I have explored the dynamics of P-element expression and splicing through state-of-the-art sequencing (long-read sequencing technology) and imaging (singlevi molecule Fluorescence In Situ Hybridisation) techniques, targeted screening of candidate regulatory proteins, and devising a genetic toolkit based on the dead-Cas9 system to definitively test the role of different factors on P-element splicing regulation in vivo. This led me to dissect the relationship between transcriptional start site and splicing regulation, as well as to uncover the regulation of antisense P-element transcripts and apparent differences in poly(A) strength within the element. In parallel, confocal imaging and analysis of P-element RNA transcripts uncovered a new facet of regulation visible through differential transcript subcellular localisation. Genetic screening of candidate regulators led to the identification of a new factor required for suppression of transposon activity, Megator. Finally, I established a transgenic system to recruit specific factors to a P-element splicing reporter to test their sufficiency in regulating splicing. Altogether, my work highlights several aspects of P-element regulation that were not previously characterised or contrast with our existing understanding of the process. It also provides evidence of novel aspects of transposon regulation by the piRNA pathway, including regulation of transcript localisation and the nuclear pore protein Megator as a putative piRNA pathway component. These results open further avenues for future work to better understand piRNA-mediated regulation of transposons. University of Cambridge 2024-06-28 Thesis Doctoral eng https://www.repository.cam.ac.uk/handle/1810/381587 embargo 2026-03-18 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/94cec854-3559-421a-8679-382b780d3c5c/download 63cc719c99030a50f140e1106abe0cb9 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/339992f5-3399-4f8d-8e59-5fcbadcf66c8/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved P-element transposon transposable element splicing germline Drosophila piRNA PIWI-interacting RNA mRNA processing mRNA localisation