University of Cambridge
Evolutionary conservation of unistrand piRNA clusters and characterisation of the piRNA precursor export machinery in Drosophila
Abstract
dc:description.abstractThe PIWI-interacting RNA (piRNA) pathway prevents endogenous genomic parasites, transposable elements, from damaging the genetic material of animal gonadal cells. Specific regions in the genome, called piRNA clusters, are thought to define each species’ piRNA repertoire and therefore its capacity to recognize and silence specific transposon families. The *Drosophila melanogaster* ovary contains two distinct types of piRNA clusters: unistrand and dual-strand clusters. While there is only one major unistrand cluster, dual-strand clusters are plentiful with more than 100 reported to be active. The major unistrand cluster *flamenco* (*flam*) is essential in the somatic compartment of the *Drosophila* ovary to restrict *Gypsy*-family transposons from infecting the neighbouring germ cells. Disruption of *flam* results in transposon de-repression and sterility, yet it remains unknown whether this silencing mechanism is present more widely amongst *Drosophila* species. During my PhD we found that unistrand, *flamenco*-like, clusters are a conserved feature across the *Drosophila* genus. Importantly, not only did we illustrate that *flamenco* itself is conserved via synteny beyond the *melanogaster* species subgroup, but through systematic characterisation of 119 *Drosophila* species an additional five distinct and entirely new clusters with *flamenco*-like characteristics were found in species that diverged from *D. melanogaster* up to 45 million years ago. Remarkably, through bioinformatic approaches and deep sequencing of somatic piRNAs, me and my colleagues showed that these loci act as bona-fide unistrand piRNA clusters and are specifically organised to selectively silence *Gypsy*-family transposons, a class of endogenous retroviruses, in the soma. Notable, these transposons are able to invade germ cells from the surrounding somatic tissue and our study therefore provides strong evidence of a function of these clusters in transposon control, similar to *flamenco*. Together, this study provides important insight into the co-evolution between virus-like *Gypsy*-family transposons and a host defence mechanism. The other type of piRNA producing loci, dual-strand clusters, are non-canonically transcribed loci requiring specialised machinery for both their transcription and nuclear export. Their export process hinges on the nuclear export factor Nxf3, its co-factor Nxt1, and the orphan protein Bootlegger (Boot). Through interaction studies, I established that Boot and Nxf3 are multivalent interaction partners. Orthogonally, XL-MS data indicates that there are interacting residues between these two proteins throughout their entire protein length. In addition, by means of recombinant protein complex purification I showed that the association of the ATP-dependent RNA helicase UAP56 with Boot is required to form a stable export complex comprising of Boot, Nxf3, UAP56 and Nxt1. At the same time, I showed that the interaction of Boot with the chromatin bound protein Deadlock prevents binding to Nxf3, thus the export complex appears to form when Boot has dislodged from the chromatin bound piRNA transcription machinery. Furthermore, mapping of the cross-linked residues of UAP56 onto the structure embedded in the human transcription export (TREX) complex revealed that all residues are located on the surface and are facing away from the TREX complex, thus possibly allowing an interaction between Boot-Nxf3 and UAP56 whilst in complex with TREX. Lastly, preliminary low-resolution structure reconstruction from Cryo-EM images, performed by our collaborator, suggests that the export complex forms a hand-like structure. Overall, this work has laid a basis for in-detail structural and biochemical characterisation of the export complex. In summary, my research improved our understanding of how this complex facilitates the export of dual-strand cluster transcripts.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2023
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- van Lopik, Jasper
- Advisor dc:contributor.advisor
-
- Nicholson, Benjamin
Subjects
dc:subject × 7Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.110462
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/371203