Elucidating the role of macromolecular complexes in vivo
Lead Research Organisation:
University of Cambridge
Department Name: Zoology
Abstract
PhD project strategic theme: Understanding the rules of life
Localised mRNA translation is a widespread mechanism for the control of gene expression, enabling cells to regulate the production of proteins in space and time. An example of this is through the sequestering of mRNA into Processing-bodies (P-bodies). P-bodies are biomolecular condensates that form from liquid-liquid phase separation of RNA and protein, allowing for the concentration of specific molecules without the encapsulating membrane. This allows for rapid responses to changing cellular conditions. P-bodies act as subcellular compartments that control the fate of cytosolic mRNAs. P-bodies and their components are implicated in a variety of human diseases (including neurodegeneration and cancer), understanding P-bodies at the molecular level may offer insight into human disease.
P-bodies act as translational regulators, but how this occurs is unknown. It is plausible that P-bodies function as a store for translationally repressed mRNA and/or sites of mRNA degradation based on the composition of their resident enzymes.
To explore the role of P-bodies in vivo I will use Drosophila as a model organism, exploiting its advantages for sophisticated genetics and live imaging. Drosophila research has identified thousands of genes with human homologues and has provided key insights into many biological processes. My use of the egg chamber and early embryo has many experimental benefits. These include being an in vivo developing system, ease of physical manipulation and providing ample experimental material.
P-bodies in Drosophila oocytes primarily function in storage and regulation of maternal mRNAs. For instance, translational control of bicoid mRNA by P-bodies in oocytes is achieved by sequestering bicoid transcripts at the core of the P-body. However, what is responsible for the targeting of bicoid mRNA to the P-body is unknown. To test potential P-body targeting sequences fly-lines with Bicoid conjugated to GFP can be generated, GFP presence would represent a read out of Bicoid translation. Flies mutant for potential P-body targeting sequences within bicoid mRNA can be generated to test the ability for these sequences to target bicoid mRNA to the P-body and thus prevent translation.
Less is known about the role of the P-bodies in the early embryo; the role of the P-bodies is hypothesised to be altered to mRNA degradation during early development. Some patterning genes present in the early embryo have mRNA with predicted half-lives of approximately 6 minutes. These are some of the shortest half-lives for mRNA in all metazoan species. Targeting of this mRNA to controlled, yet rapid to degradation machinery and could hint at the role of P-bodies in the early Drosophila embryo.
I have presented preliminary analysis using in situ hybridisation chain reaction and immunohistochemistry which assessed the co-localisation of early embryonic mRNAs with key P-body proteins. Co-localisation was high specifically with apically localised patterning genes. The early embryo could offer a novel in vivo system for addressing some key questions that exist surrounding P-bodies: how RNA is targeted to the P-bodies? How are P-bodies assembled? What is the function of P-bodies? This can be achieved through creating tagged mRNA for these genes and injecting these into Drosophila embryos.
Localised mRNA translation is a widespread mechanism for the control of gene expression, enabling cells to regulate the production of proteins in space and time. An example of this is through the sequestering of mRNA into Processing-bodies (P-bodies). P-bodies are biomolecular condensates that form from liquid-liquid phase separation of RNA and protein, allowing for the concentration of specific molecules without the encapsulating membrane. This allows for rapid responses to changing cellular conditions. P-bodies act as subcellular compartments that control the fate of cytosolic mRNAs. P-bodies and their components are implicated in a variety of human diseases (including neurodegeneration and cancer), understanding P-bodies at the molecular level may offer insight into human disease.
P-bodies act as translational regulators, but how this occurs is unknown. It is plausible that P-bodies function as a store for translationally repressed mRNA and/or sites of mRNA degradation based on the composition of their resident enzymes.
To explore the role of P-bodies in vivo I will use Drosophila as a model organism, exploiting its advantages for sophisticated genetics and live imaging. Drosophila research has identified thousands of genes with human homologues and has provided key insights into many biological processes. My use of the egg chamber and early embryo has many experimental benefits. These include being an in vivo developing system, ease of physical manipulation and providing ample experimental material.
P-bodies in Drosophila oocytes primarily function in storage and regulation of maternal mRNAs. For instance, translational control of bicoid mRNA by P-bodies in oocytes is achieved by sequestering bicoid transcripts at the core of the P-body. However, what is responsible for the targeting of bicoid mRNA to the P-body is unknown. To test potential P-body targeting sequences fly-lines with Bicoid conjugated to GFP can be generated, GFP presence would represent a read out of Bicoid translation. Flies mutant for potential P-body targeting sequences within bicoid mRNA can be generated to test the ability for these sequences to target bicoid mRNA to the P-body and thus prevent translation.
Less is known about the role of the P-bodies in the early embryo; the role of the P-bodies is hypothesised to be altered to mRNA degradation during early development. Some patterning genes present in the early embryo have mRNA with predicted half-lives of approximately 6 minutes. These are some of the shortest half-lives for mRNA in all metazoan species. Targeting of this mRNA to controlled, yet rapid to degradation machinery and could hint at the role of P-bodies in the early Drosophila embryo.
I have presented preliminary analysis using in situ hybridisation chain reaction and immunohistochemistry which assessed the co-localisation of early embryonic mRNAs with key P-body proteins. Co-localisation was high specifically with apically localised patterning genes. The early embryo could offer a novel in vivo system for addressing some key questions that exist surrounding P-bodies: how RNA is targeted to the P-bodies? How are P-bodies assembled? What is the function of P-bodies? This can be achieved through creating tagged mRNA for these genes and injecting these into Drosophila embryos.
Organisations
People |
ORCID iD |
| Timothy Thomas Weil (Primary Supervisor) | |
| Elise Wilby (Student) |
