{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/372286"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/372286","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Investigating the function and mechanistic control of processing bodies in early Drosophila melanogaster development","abstract":"Biomolecular condensates provide one mechanism by which cells can organise their internal environment. These cellular compartments are described as a mesoscopic concentration of biological macromolecules that are physically segregated from the rest of the cytoplasm or nucleoplasm, despite the lack of a limiting membrane. This results in a specialised intracellular region that likely carries out a specific cellular process. A particularly well-studied class of biomolecular condensates that contain both proteins and RNAs are the ribonucleoprotein (RNP) granules. These granules have been described in many cell types and have been assigned numerous critical functions in RNA metabolism. However, recently, some have argued that the ability of multiple individual mRNP complexes to condense into granules may not always provide additional function beyond the formation of single mRNP complexes. In this thesis, I utilise the genetically, chemically, and physically manipulatable model system of *Drosophila melanogaster* oogenesis and early embryogenesis to investigate the function and mechanistic control of processing bodies (P-bodies), an evolutionarily conserved RNP granule. As perceived sites of RNA metabolism, the function of P-bodies, if any, has been widely debated. I investigate P-bodies, observing and manipulating their relationship with axis-patterning RNA to explore how this affects its post-transcriptional regulation. I show that P-bodies become microscopically visible in early oogenesis and increase their size and association with the anterior determinant, *bicoid* (*bcd*) mRNA, throughout oogenesis. When the oocyte reaches maturity, virtually all *bcd* is found in P-bodies. At this point in developmental time, *bcd* is tightly localised at the anterior margin, translationally repressed, and stable for long periods of time. To understand the function of these RNP granules in the post-transcriptional regulation of *bcd*, it was critical to be able to manipulate the P-bodies *in vivo*. Therefore, I performed a small-scale genetic screen of known P-body components to investigate which proteins affected P-body formation, integrity, and morphology. From this data, I further investigated several critical components that either affected the ability of P-body mRNPs to condense into larger bodies or altered the physical properties of P-bodies, which in turn affected their ability to retain *bcd*. In mature oocytes, when *bcd* fails to localise or condense into P-bodies either through genetic or chemical manipulation, I show several critical changes to its post-transcriptional regulation. Most notably, Bcd protein translation can be detected, *bcd* is no longer closely localised to the anterior margin, and the long-term stability of *bcd* mRNA is reduced. Together, this suggests that the localisation of *bcd* to P-bodies themselves rather than the function of individual mRNP complexes is critical for several aspects of RNA regulation. At the oocyte-to-embryo transition, many important cellular changes occur, resulting in the dispersal of P-bodies and the release of their RNA. I investigated the mechanisms behind this process, highlighting the importance of the RNA release from P-bodies for its correct temporal translation. I identified the downstream events of the calcium transient, most probably calcium-dependent post-translational modifications (PTMs), as being important for this release of RNA from P-bodies and its subsequent translation. I also investigate the reformation of P-bodies in early embryogenesis, showing a re-association of *bcd* with the newly formed P-bodies coinciding with the developmental timing of their degradation. This adds to the growing body of evidence that P-bodies in *Drosophila* embryos can also act as degradation hubs. This further highlights that these RNP granules are changing and dynamic entities whose function depends highly on the cellular context, emphasising the importance of *in vivo* work to understand biomolecular condensates. Taken together, this novel data shows multiple context-dependent biological functions for P-bodies in the post-transcriptional regulation of RNA in living cells.","abstract_html":"Biomolecular condensates provide one mechanism by which cells can organise their internal environment. These cellular compartments are described as a mesoscopic concentration of biological macromolecules that are physically segregated from the rest of the cytoplasm or nucleoplasm, despite the lack of a limiting membrane. This results in a specialised intracellular region that likely carries out a specific cellular process. A particularly well-studied class of biomolecular condensates that contain both proteins and RNAs are the ribonucleoprotein (RNP) granules. These granules have been described in many cell types and have been assigned numerous critical functions in RNA metabolism. However, recently, some have argued that the ability of multiple individual mRNP complexes to condense into granules may not always provide additional function beyond the formation of single mRNP complexes. In this thesis, I utilise the genetically, chemically, and physically manipulatable model system of *Drosophila melanogaster* oogenesis and early embryogenesis to investigate the function and mechanistic control of processing bodies (P-bodies), an evolutionarily conserved RNP granule. As perceived sites of RNA metabolism, the function of P-bodies, if any, has been widely debated. I investigate P-bodies, observing and manipulating their relationship with axis-patterning RNA to explore how this affects its post-transcriptional regulation. I show that P-bodies become microscopically visible in early oogenesis and increase their size and association with the anterior determinant, *bicoid* (*bcd*) mRNA, throughout oogenesis. When the oocyte reaches maturity, virtually all *bcd* is found in P-bodies. At this point in developmental time, *bcd* is tightly localised at the anterior margin, translationally repressed, and stable for long periods of time. To understand the function of these RNP granules in the post-transcriptional regulation of *bcd*, it was critical to be able to manipulate the P-bodies *in vivo*. Therefore, I performed a small-scale genetic screen of known P-body components to investigate which proteins affected P-body formation, integrity, and morphology. From this data, I further investigated several critical components that either affected the ability of P-body mRNPs to condense into larger bodies or altered the physical properties of P-bodies, which in turn affected their ability to retain *bcd*. In mature oocytes, when *bcd* fails to localise or condense into P-bodies either through genetic or chemical manipulation, I show several critical changes to its post-transcriptional regulation. Most notably, Bcd protein translation can be detected, *bcd* is no longer closely localised to the anterior margin, and the long-term stability of *bcd* mRNA is reduced. Together, this suggests that the localisation of *bcd* to P-bodies themselves rather than the function of individual mRNP complexes is critical for several aspects of RNA regulation. At the oocyte-to-embryo transition, many important cellular changes occur, resulting in the dispersal of P-bodies and the release of their RNA. I investigated the mechanisms behind this process, highlighting the importance of the RNA release from P-bodies for its correct temporal translation. I identified the downstream events of the calcium transient, most probably calcium-dependent post-translational modifications (PTMs), as being important for this release of RNA from P-bodies and its subsequent translation. I also investigate the reformation of P-bodies in early embryogenesis, showing a re-association of *bcd* with the newly formed P-bodies coinciding with the developmental timing of their degradation. This adds to the growing body of evidence that P-bodies in *Drosophila* embryos can also act as degradation hubs. This further highlights that these RNP granules are changing and dynamic entities whose function depends highly on the cellular context, emphasising the importance of *in vivo* work to understand biomolecular condensates. Taken together, this novel data shows multiple context-dependent biological functions for P-bodies in the post-transcriptional regulation of RNA in living cells.","abstract_has_math":false,"creators":["Wilby, Elise"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Weil, Timothy"],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-12-31","date_published":"2023-12-31","updated_at":"2026-07-22T22:24:20Z","subjects":["Biomolecular Condensates","Drosophila","Drosophila oogenesis","Processing bodies","RNP granules","Translational Control"],"languages":["eng"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/879d15a5-616d-48ae-8354-d583b8414ea8/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.111156","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Weil, Timothy"]},{"key":"dc:creator","label":"Author","values":["Wilby, Elise"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2023-12-31"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/372286"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Biomolecular Condensates","Drosophila","Drosophila oogenesis","Processing bodies","RNP granules","Translational Control"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/879d15a5-616d-48ae-8354-d583b8414ea8/download","https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.111156"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/481cad54-ca94-45a4-94be-65d0624c116f/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Biomolecular condensates provide one mechanism by which cells can organise their internal environment. 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In this thesis, I utilise the genetically, chemically, and physically manipulatable model system of *Drosophila melanogaster* oogenesis and early embryogenesis to investigate the function and mechanistic control of processing bodies (P-bodies), an evolutionarily conserved RNP granule. As perceived sites of RNA metabolism, the function of P-bodies, if any, has been widely debated. I investigate P-bodies, observing and manipulating their relationship with axis-patterning RNA to explore how this affects its post-transcriptional regulation. I show that P-bodies become microscopically visible in early oogenesis and increase their size and association with the anterior determinant, *bicoid* (*bcd*) mRNA, throughout oogenesis. When the oocyte reaches maturity, virtually all *bcd* is found in P-bodies. At this point in developmental time, *bcd* is tightly localised at the anterior margin, translationally repressed, and stable for long periods of time. To understand the function of these RNP granules in the post-transcriptional regulation of *bcd*, it was critical to be able to manipulate the P-bodies *in vivo*. Therefore, I performed a small-scale genetic screen of known P-body components to investigate which proteins affected P-body formation, integrity, and morphology. From this data, I further investigated several critical components that either affected the ability of P-body mRNPs to condense into larger bodies or altered the physical properties of P-bodies, which in turn affected their ability to retain *bcd*. In mature oocytes, when *bcd* fails to localise or condense into P-bodies either through genetic or chemical manipulation, I show several critical changes to its post-transcriptional regulation. Most notably, Bcd protein translation can be detected, *bcd* is no longer closely localised to the anterior margin, and the long-term stability of *bcd* mRNA is reduced. Together, this suggests that the localisation of *bcd* to P-bodies themselves rather than the function of individual mRNP complexes is critical for several aspects of RNA regulation. At the oocyte-to-embryo transition, many important cellular changes occur, resulting in the dispersal of P-bodies and the release of their RNA. I investigated the mechanisms behind this process, highlighting the importance of the RNA release from P-bodies for its correct temporal translation. I identified the downstream events of the calcium transient, most probably calcium-dependent post-translational modifications (PTMs), as being important for this release of RNA from P-bodies and its subsequent translation. I also investigate the reformation of P-bodies in early embryogenesis, showing a re-association of *bcd* with the newly formed P-bodies coinciding with the developmental timing of their degradation. This adds to the growing body of evidence that P-bodies in *Drosophila* embryos can also act as degradation hubs. This further highlights that these RNP granules are changing and dynamic entities whose function depends highly on the cellular context, emphasising the importance of *in vivo* work to understand biomolecular condensates. 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This results in a specialised intracellular region that likely carries out a specific cellular process. A particularly well-studied class of biomolecular condensates that contain both proteins and RNAs are the ribonucleoprotein (RNP) granules. These granules have been described in many cell types and have been assigned numerous critical functions in RNA metabolism. However, recently, some have argued that the ability of multiple individual mRNP complexes to condense into granules may not always provide additional function beyond the formation of single mRNP complexes. In this thesis, I utilise the genetically, chemically, and physically manipulatable model system of *Drosophila melanogaster* oogenesis and early embryogenesis to investigate the function and mechanistic control of processing bodies (P-bodies), an evolutionarily conserved RNP granule. As perceived sites of RNA metabolism, the function of P-bodies, if any, has been widely debated. I investigate P-bodies, observing and manipulating their relationship with axis-patterning RNA to explore how this affects its post-transcriptional regulation. I show that P-bodies become microscopically visible in early oogenesis and increase their size and association with the anterior determinant, *bicoid* (*bcd*) mRNA, throughout oogenesis. When the oocyte reaches maturity, virtually all *bcd* is found in P-bodies. At this point in developmental time, *bcd* is tightly localised at the anterior margin, translationally repressed, and stable for long periods of time. To understand the function of these RNP granules in the post-transcriptional regulation of *bcd*, it was critical to be able to manipulate the P-bodies *in vivo*. Therefore, I performed a small-scale genetic screen of known P-body components to investigate which proteins affected P-body formation, integrity, and morphology. From this data, I further investigated several critical components that either affected the ability of P-body mRNPs to condense into larger bodies or altered the physical properties of P-bodies, which in turn affected their ability to retain *bcd*. In mature oocytes, when *bcd* fails to localise or condense into P-bodies either through genetic or chemical manipulation, I show several critical changes to its post-transcriptional regulation. Most notably, Bcd protein translation can be detected, *bcd* is no longer closely localised to the anterior margin, and the long-term stability of *bcd* mRNA is reduced. Together, this suggests that the localisation of *bcd* to P-bodies themselves rather than the function of individual mRNP complexes is critical for several aspects of RNA regulation. At the oocyte-to-embryo transition, many important cellular changes occur, resulting in the dispersal of P-bodies and the release of their RNA. I investigated the mechanisms behind this process, highlighting the importance of the RNA release from P-bodies for its correct temporal translation. I identified the downstream events of the calcium transient, most probably calcium-dependent post-translational modifications (PTMs), as being important for this release of RNA from P-bodies and its subsequent translation. I also investigate the reformation of P-bodies in early embryogenesis, showing a re-association of *bcd* with the newly formed P-bodies coinciding with the developmental timing of their degradation. This adds to the growing body of evidence that P-bodies in *Drosophila* embryos can also act as degradation hubs. This further highlights that these RNP granules are changing and dynamic entities whose function depends highly on the cellular context, emphasising the importance of *in vivo* work to understand biomolecular condensates. 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