Analysis of the role of ribonucleases in the regulation of epithelial sheet sealing
Lead Research Organisation:
University of Sussex
Department Name: Brighton and Sussex Medical School
Abstract
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Technical Summary
Ribonucleases have been well studied in yeast and bacteria, but their biological significance to multicellular organisms is not well understood. However, there is increasing evidence that specific, timed transcript degradation is critical for many cellular processes, including early development, RNA interference and nonsense-mediated decay. We have used a reverse genetics approach in Drosophila to show that mutations in the 5 prime-3 prime exoribonuclease pacman result in severe defects in thorax closure such that mutants have clefts along the back of the thorax. The dorsal part of the thorax in wild-type Drosophila is formed by cells at the leading edges of the two wing imaginal discs which stretch, migrate towards each other and then fuse along the dorsal midline. This epithelial cell movement is very similar to seen in dorsal closure in Drosophila, hind-brain closure in vertebrates, ventral enclosure in C. elegans and wound healing in humans. Our preliminary experiments have shown that pacman genetically interacts with puckered, a conserved member of the JNK signalling pathway, which is known to be involved in thoracic closure. We have also shown that pacman protein is localised in cytoplasmic foci, which are likely to be specific sites of 5 prime-3 prime mRNA degradation (P-bodies). We propose that pacman plays a key role in epithelial sheet sealing, by targeting particular RNAs. To test this hypothesis we will first use genetic, molecular biology and micro-array analysis to establish whether puckered, or another member of the JNK pathway, is a specific target of the pacman ribonuclease. We will then construct transgenic flies to determine which regions of the target RNA are recognised by the 5 prime-3 prime degradation machinery. Second, we will use confocal microscopy to investigate the intra-cellular localisation of PACMAN to determine whether it is associated with the actin cytoskeleton or other cellular components. Third, we will examine the localisation of PACMAN in relation to other proteins in the 5 prime-3 prime degradation pathway, to determine, as our pilot data suggests, whether PACMAN has a key role in the formation of P-bodies. Finally, we will determine the involvement of PACMAN in wound healing in Drosophila by comparing the wound healing process in mutant, compared to wild-type flies. These innovative studies will shed light on the role of pacman ribonuclease in development.
Organisations
Publications
Grima DP
(2008)
The 5'-3' exoribonuclease pacman is required for epithelial sheet sealing in Drosophila and genetically interacts with the phosphatase puckered.
in Biology of the cell
Lin MD
(2008)
Drosophila processing bodies in oogenesis.
in Developmental biology
Zabolotskaya MV
(2008)
The 5'-3' exoribonuclease Pacman is required for normal male fertility and is dynamically localized in cytoplasmic particles in Drosophila testis cells.
in The Biochemical journal
Description | The aim of the project was to investigate a fundamental and novel mechanism of gene regulation which is applicable to all eukaryotes. The process to be investigated was the control of RNA stability, which is the least understood mechanism of gene regulation in multi-cellular organisms. It is now well established that biological processes require carefully controlled systems to maintain a finely tuned balance between production and destruction. For example, at the cellular level, proliferation and apoptosis are tightly regulated to maintain the correct cell numbers; at the protein level the amounts of protein are carefully controlled by the rate of translation and the rate of degradation by the proteasome. In a similar way, levels of RNA (which often determine the levels of protein expressed) are a balance between transcription and degradation. Until relatively recently, it was generally assumed that RNA degradation was a relatively constitutive process. However, recent work by my group and others has shown that it is highly regulated, varies during development or in response to environmental factors and that specific RNAs can be targeted for degradation. The key findings of the projects are as follows: 1. Our work has started to reveal the molecular mechanisms whereby the 5' - 3' exoribonuclease pacman/xrn-1 regulates epithelial sheet sealing, which is a process whereby two sheets of cells stretch, move together and fuse to form a continuous epithelium. Using genetic and molecular biological approaches, we have shown that mutations in pacman result in up-regulation of members of the conserved JNK pathway. 2. We have shown that Pacman is localised in particles in the amnioserosa, a tissue which underlies the moving epithelial sheets. This unexpected result has allowed us to formulate a hypothesis for the function of Pacman in cell movement. 3. We have demonstrated that pacman is required for normal wound healing in Drosophila. Interestingly, older flies (7-9 days old) heal less well than younger (3-5 day old) flies, suggesting that Drosophila may be a good model to study the effect of ageing on wound healing. In addition to the publications given elsewhere, the work of the project was presented at 9 International meetings. Resources generated. Transgenic lines. In this project, we successfully generated 20 transgenic lines which express either wild-type pacman or nuclease-dead pacman under the control of the GAL4-UAS system. These reagents were created by the first post-doc hired on the grant, Dr. Steve Hebbes, and further characterised by Dr. Dom Grima when the grant moved to Brighton and Sussex Medical School. The lines are available on request for other laboratories and will be placed in the public fly stock centres, following full verifications and stabilisation. |
Exploitation Route | This work was developed further in subsequent BBSRC grants. The work started at a time when little was known about gene regulation at the level of RNA and the work of this project helped to develop the field. |
Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
Description | The non-academic Impacts of the research carried out in this project were as follows: 1. The research was used in a collaborative project with a breast cancer clinician to investigate the relationship of wound healing and breast cancer. 2. The work was publicised at Open Days and in presentations to schoolchildren. 3. The development of the work, through subsequent BBSRC grants, has led to two biomarker patents and collaborations on microRNAs as biomarkers in myeloma, melanoma and septic shock. |
First Year Of Impact | 2005 |
Sector | Healthcare,Pharmaceuticals and Medical Biotechnology |
Description | Conference grant 2006 |
Amount | £1,160 (GBP) |
Organisation | The Royal Society |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2006 |
End | 03/2006 |
Description | Studentship bursary and consumables 2004 |
Amount | £76,000 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 10/2004 |
End | 03/2008 |
Description | Studentship bursary and consumables 2005 |
Amount | £67,888 (GBP) |
Organisation | Royal Victoria Infirmary |
Sector | Hospitals |
Country | United Kingdom |
Start | 09/2005 |
End | 08/2007 |
Description | Studentship bursary and consumables 2007 |
Amount | £76,000 (GBP) |
Organisation | University of Sussex |
Department | Brighton and Sussex Medical School |
Sector | Academic/University |
Country | United Kingdom |
Start | 10/2007 |
End | 03/2011 |
Description | Summer studentship 2005 |
Amount | £1,600 (GBP) |
Organisation | Nuffield Foundation |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 07/2005 |
End | 09/2006 |
Description | Summer studentship 2006 |
Amount | £2,000 (GBP) |
Organisation | The Genetics Society |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 06/2006 |
End | 09/2006 |
Description | Summer studentship 2007 |
Amount | £1,600 (GBP) |
Organisation | Nuffield Foundation |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 07/2007 |
End | 09/2007 |
Description | Summer studentship 2008 |
Amount | £1,600 (GBP) |
Organisation | University of Sussex |
Sector | Academic/University |
Country | United Kingdom |
Start | 07/2008 |
End | 09/2008 |
Description | Summer studentship 2008 |
Amount | £1,600 (GBP) |
Organisation | Biochemical Society |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 07/2008 |
End | 09/2008 |
Description | Summer studentship 2010 |
Amount | £1,600 (GBP) |
Organisation | University of Sussex |
Sector | Academic/University |
Country | United Kingdom |
Start | 07/2010 |
End | 09/2010 |
Description | Treaty of Windsor programme 2008 |
Amount | £1,000 (GBP) |
Organisation | British Council |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 04/2008 |
End | 04/2009 |
Description | pump priming grant 2008 |
Amount | £5,000 (GBP) |
Organisation | University of Brighton |
Sector | Academic/University |
Country | United Kingdom |
Start | 07/2008 |
End | 06/2009 |
Title | Transgenic Drosophila |
Description | Drosophila stocks expressing mutant versions of pacman. These have been made available upon request. |
Type Of Material | Model of mechanisms or symptoms - non-mammalian in vivo |
Provided To Others? | Yes |
Impact | Acknowledgements on publications |
Description | BSMS Away Days |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | The presentation sparked further collaborations with clinicians Further collaborations, particularly regarding research on microRNA biomarkers in a number of diseases, has ensued. |
Year(s) Of Engagement Activity | 2007,2010,2011,2012,2013 |
Description | Open Days |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Increase in understanding of laboratory-based research. Increase in applications for Medical School |
Year(s) Of Engagement Activity | Pre-2006,2006,2007,2008,2009,2010,2011,2012,2013,2014 |
Description | Tissue Regeneration Sandpit 2007 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | Together with two other researchers, I organised a Sandpit event with the aim of forging collaborations between Academics, clinicians and industrialists. A number of collaborations were set up as a result of this event. A number of collaborations were set up as a result of this event. My team won a start-up grant of £5000. |
Year(s) Of Engagement Activity | 2007 |