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Title: Mechanisms of piRNA biogenesis and co-transcriptional silencing of transposable elements in Drosophila
Author: Munafo, Marzia
ISNI:       0000 0004 9348 1789
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
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A large fraction of eukaryotic genomes consists of mobile, repetitive elements called transposons. Since their uncontrolled mobilisation is a potentially harmful event, several molecular mechanisms have evolved to counteract transposon activation and thus safeguard genome integrity. Among these is the piRNA pathway, a gonad-specific system based on small non-coding RNAs that recognise active transposons and instruct their silencing. Ultimately, piRNAs trigger epigenetic silencing of transposon loci. The work in this thesis investigates the molecular mechanisms by which piRNAs are produced from the correct substrates and how piRNAs can induce silencing of target loci in Drosophila melanogaster. First, I investigated how piRNA precursors are selected for processing and how they are transported to mitochondria, where piRNA production occurs. I explored the role of an uncharacterised Drosophila gene previously implicated in germline transposon control: CG10880/Daedalus (Daed). I found that Daed is an essential component of the mitochondrial piRNA biogenesis machinery and that it recruits the RNA helicase Armitage (Armi) to mitochondria. If Armi fails to be recruited, piRNA biogenesis cannot occur since Armi’s role is that of delivering piRNA precursors to the mitochondrial processing machinery. Secondly, I investigated how the major piRNA precursor in somatic cells, namely flamenco (flam) transcript, is exported and specified for downstream piRNA production. I uncovered that flam export is closely linked to the assembly of peri-nuclear condensates of the helicase fs(1)Yb (Yb). Furthermore, some subunits of the Nuclear Pore Complex (NPC) are also required for the production flam-derived piRNAs, thus suggesting the evolution of a specialised machinery that couples nuclear export and processing of this transcript. Finally, I set out to understand how piRNAs trigger silencing of active transposons. I found that Panoramix (Panx), the central effector of piRNA-guided epigenetic silencing, assembles into a complex with two other proteins: Nxf2 and Nxt1. We characterised the dependencies within the complex and found that all three components are essential to initiate silencing. Intriguingly, Nxf2 and Nxt1 belong to the family of nuclear export factors, thus suggesting that the piRNA pathway has co-opted proteins involved in RNA export and repurposed them for transposon control. Overall, this work provides new insights on the molecular mechanisms of piRNA-guided transposon silencing in Drosophila and shows evidence that transposon control pathways can exploit cellular factors for novel functions.
Supervisor: Hannon, Gregory J. ; Czech, Benjamin Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: piwi ; piRNA ; transposons