Function of the exoribonuclease pacman in cell movement and cell shape change

Lead Research Organisation: University of Sussex
Department Name: Brighton and Sussex Medical School

Abstract

Wound healing requires the surrounding skin cells to stretch, draw together and seal to cover the hole in the layer of skin cells. This process of wound healing is similar to other cell sheet movements in the development of all embryos. We have shown that an enzyme involved in the destruction of messenger RNAs is necessary for the cell sheet sealing process in the fruit fly Drosophila melanogaster. This enzyme, which is named Pacman, chews up these messenger RNAs from one end to the other. Messenger RNAs are the molecules which instruct the cell to make particular proteins. We have found out that Pacman is likely to be part of a large complex of proteins, a 'Death Star' which can efficiently destroy or imprison messenger RNAs. We suspect that Pacman must destroy RNAs that would potentially stop the cell sheet sealing process. The aim of this project is to find out how Pacman can control this hole closure process. The control mechanism probably involves targeting particular mRNAs for destruction by the 'Death Star' complex and we will test whether this is the case. Although this project will be carried out using the fruit fly Drosophila, it also has relevance for epithelial sheet sealing processes such as wound healing in humans as the cellular processes are surprisingly similar in both organisms. In addition, the Pacman enzyme is extremely similar between flies and humans therefore the insights we gain during this project may help us to improve the treatment for wounds and also help us to understand the cell movement process in embryos. The work described in this proposal is also directed towards the general question of gene regulation at the RNA level. It has recently become recognised that genes can be controlled at the level of the messenger RNA by using RNA itself as a tool to turn down specific genes. In a process termed RNA interference, it is possible to downregulate any particular RNA in the cell. Since Pacman and its homologues have been shown to be involved in RNA interference, this project will also may also provide innovative insights into the improval of this process for therapeutic purposes.

Technical Summary

The conserved 5' - 3' exoribonuclease Pacman/Xrn1 is known to play a critical role in gene regulatory events such as control of mRNA stability, RNA interference, nonsense-mediated decay and regulation via microRNAs. We have shown that this enzyme is often located in cytoplasmic particles termed P-bodies which are thought to be involved in the translational repression and degradation of RNAs. Our recent results show that mutations in the exoribonuclease pacman lead to defects in epithelial sheet sealing processes such as dorsal/thorax closure and wound healing in Drosophila. These epithelial sheet sealing processes are morphologically similar to those seen in eyelid fusion and hind-brain closure in mammals. The overall aim of the project is to understand how the Drosophila exoribonuclease Pacman regulates dorsal closure. We propose that pacman plays a key role in this process by targeting particular RNAs. An attractive hypothesis is that pacman controls dorsal closure by regulating the conserved JNK signaling pathway. This cell signaling pathway is well known to orchestrate cell movement, cell shape change and cell adhesion during the dorsal closure process. To determine the function of pacman in dorsal closure, we will analyse in detail the phenotypic consequences of pacman mutations on dorsal closure and on known regulators of the dorsal closure process. This analysis will give information on the cellular pathways targeted by pacman. We will also identify targets of Pacman during dorsal closure using biochemical techniques and micro-array analysis. The localisation of Pacman protein in relation to P-body components and translational repressors will also be examined in order to assess the involvement of translational repression in this process. These innovative studies will shed light on the role of the pacman ribonuclease in dorsal closure and, more generally, help us to understand the mechanisms whereby particular RNAs are targeted for degradation.
 
Description Development of an organism from egg to adult requires sets of genes to be switched on and off at particular times and in the correct order. If genes are not switched off when necessary, cells can continue to multiply in an uncontrolled way leading to cancer. As well as being important in cancer, gene regulation is crucial in controlling the growth, differentiation and cell movements which are required to form the particular cells and tissues in the body. Since growth, differentiation and cell movement are important in cancer, the understanding of gene control which affects these processes is crucial. Therefore studying the mechanisms whereby genes are switched off (as well as on) is vitally important for providing basic knowledge that has potential to lead to novel therapeutics.

The current work has examined a gene called pacman, which encodes an enzyme involved in the destruction of messenger RNAs. This enzyme chews up these messenger RNAs from one end to the other. Messenger RNAs are the molecules which instruct the cell to make particular proteins. We have found out that Pacman is part of a large complex of proteins, a Death Star which can efficiently destroy or imprison messenger RNAs. Using the fruit fly Drosophila as a model organism, our previous work had shown that pacman is required for normal development, particularly during processes where sheets or tissues need to grow and move together. In this project, we concentrated on the tissues known as imaginal discs, which form adult structures such as wings and legs. These imaginal discs are excellent model systems to study growth, differentiation and cell movement.

By examining the imaginal discs of pacman mutants, we found that they were much smaller than normal. This suggested to us that pacman might primarily be involved in cell proliferation and differentiation. To test this we used the genetic tools that are available in Drosophila to knock down pacman in parts of the wing imaginal discs and observe the effects on the wings and thorax of the adult fly. In all cases we found that knockdown of pacman was severely detrimental, resulting in lack of growth of that part of the tissue. This suggested that Pacman is normally involved in cell proliferation, either by promoting cell division, increasing cell size, or inhibiting cell death.

Our next aim was to understand how Pacman controls particular sets of genes involved in proliferation. Using a method termed microarray analysis, we showed that Pacman normally affects an RNA encoding a protein called Simjang (Korean for strong heart). This protein has been shown by other researchers to control a gene silencing complex (the NuRD complex) which shuts down parts of the chromosome, preventing genes from being turned on.

This gene silencing complex is important because it is known to be involved in many critical cellular events including ageing, tissue regeneration, formation of blood cells, and spread of cancer cells. This is the first time that an enzyme involved in degradation of messenger RNA has been shown to be directly affecting a gene silencing complex. The mechanism of gene regulation we have discovered is entirely novel; as yet no research group has found this link between RNA degradation and gene silencing. Since all the proteins involved are similar in Drosophila and humans the new control modulewe have discovered is likely to be relevant to gene regulation in humans. Since Pacman is known to be important in other important cellular events such as stem cell function and fertility, this work may also shed light on the molecular mechanisms of these processes in other tissues. Therefore the insights we gain during this project may help us to improve treatment for cancer and other diseases and also help us to understand the ways that tissues grow and develop. This project will therefore provide valuable insights into a new method of gene regulation which can be used in the
development of new therapeutics.

Resources generated.
The following resources were generated:

1. Drosophila stocks expressing mutant versions of pacman. These have been made available upon request.

2. Microarray data comparing the expression of RNAs across the whole genome in pcm5 and isogenic controls.

Conference proceedings: 22
Exploitation Route The expertise gained during this project has allowed a collaboration with a colleague in the Medical School, Dr Tim Chevassut, a consultant haematologist working on blood cancers. Together, using blood samples from patients, we have developed a novel miRNA biomarker which is diagnostic for myeloma and its precancerous state (MGUS). The novelty of
this biomarker is that it is minimally invasive in that it uses serum from circulating blood. We have patented our miRNA signature for circulating miRNAs in MGUS/myeloma through the University of Sussex. The Sussex Innovation office have marketed our new biomarker assay in order to attract Industrial Companies with a view to developing this biomarker commercially. To date, we are in advanced negotiations with a number of Industrial companies who wish to exploit this work. We hope to work together with these companies in order to commercialise these biomarkers.



In addition to the above Industrial connection, we have also set up a collaboration with the Blond McIndoe Research Foundation. The Blond McIndoe Research Foundation are interested in wound healing as they have expertise in plastic surgery and treatment of melanoma. This collaboration has led to a formal collaboration with a joint post-doc funded by a charitable donation from the Myfanwy Townsend Trust. The post-doc will spend part of her time in my lab, identifying microRNAs biomarkers involved in the progression of melanoma.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology
 
Description Communications and engagement (a) New collaborative links: As the project has been carried out in a Medical School, there has been ample opportunity for interaction with Medical Colleagues in collaborative research. This has already resulted in a collaboration with Dr. Tim Chevassut, a consultant haematologist, in a joint project and 2 joint grant applications. Our project combines the clinical expertise of my clinical colleague with that of my lab's expertise in working with RNA. We have already made progress in developing a novel biomarker for certain types of leukaemias and have now patented a novel miRNA signature for myeloma. (b)Engagement and communication. We have been involved in a number of events to ensure good engagement and communication. I organised a "Sandpit" on Tissue Regeneration, "Wound healing and Stem cells" which brought together researchers from BSMS, University of Brighton, University of Sussex and clinicians from Brighton and Sussex University Hospitals in collaborative discussions. I was also invited to participate in the University of Sussex "Curious" event where local representatives from local businesses and other stakeholders were able to interact. (c) New networking groups. I recently set up the South Coast RNA Network meetings, which involve groups working on RNA Biology from Universities on (or near) the South Coast of England together with representatives from Industry. The first meeting, in November 2009, was organised by myself, Professor Simon Morley and the University of Sussex Business and Enterprize Office. The half-day meeting, which was free to Academic delegates, was extremely successful and has been followed up by a second meeting in November 2010 at the University of Surrey, a 3rd meeting in November 2011 at the University of Portsmouth and a subsequent meeting planned at the University of Kent. The co-organisers for these meetings are post-docs and PhD students, which gave them an opportunity to learn the skills of meeting organisation. (d) Websites and interactive media. The work of my group has frequently been chosen by the BSMS and University of Brighton public engagement offices to be published in a way that is accessible to the general audience. Research news, together with brief explanations of research in layman's language, has been regularly published on the BSMS Website. (e) Schools and public engagement I regularly host a school student in my lab to introduce them to Medical Research. We have also been visited by groups of younger children, e.g. from the Dorothy Stringer School to show them the kind of research that we do in our labs. In addition, groups of parents are invited for lab tours during the open days for Medical Students which take place several times each year.
First Year Of Impact 2008
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic
 
Description Studentship bursary and consumables 2012 (BPT)
Amount £77,400 (GBP)
Organisation University of Sussex 
Department Brighton and Sussex Medical School
Sector Academic/University
Country United Kingdom
Start 10/2012 
End 09/2016
 
Description Studentship bursary and consumables 2012 (SR)
Amount £77,400 (GBP)
Organisation University of Sussex 
Department Brighton and Sussex Medical School
Sector Academic/University
Country United Kingdom
Start 10/2012 
End 09/2016
 
Description Summer Studentship 2011
Amount £1,600 (GBP)
Organisation Nuffield Foundation 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2011 
End 09/2011
 
Description Summer Studentship 2012
Amount £1,600 (GBP)
Organisation Biochemical Society 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2012 
End 09/2012
 
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 2008
Amount £1,600 (GBP)
Organisation University of Sussex 
Sector Academic/University
Country United Kingdom
Start 07/2008 
End 09/2008
 
Description Summer studentship 2009
Amount £2,550 (GBP)
Organisation The Genetics Society 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2009 
End 09/2009
 
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 Summer studentship 2010
Amount £1,200 (GBP)
Organisation Biochemical Society 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2010 
End 09/2010
 
Description Summer studentship 2012
Amount £1,600 (GBP)
Organisation Biochemical Society 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2012 
End 09/2012
 
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 and Dis3L2. These have been made available upon request or sent to the Bloomington stock centre for distribution upon request. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2013 
Provided To Others? Yes  
Impact Acknowledgements on publications 
 
Description Blond McIndoe Research Foundation 
Organisation Blond McIndoe Research Foundation
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution A post-doc, funded by the Blond McIndoe Research Foundation, worked in my lab for 8 months. Myself and my research team taught and advised her on techniques. We also provided some consumables e.g. plasticware and the use of our equipment such as the Bioanalyser and MiSeq machine. Subsequent to that we were awarded a BBSRC Sparking Impact Award entitled Investigating the potential of microRNAs as biomarkers in malignant melanoma to fund a post-doc, consumables, patent searches and market surveys. The postdoc, Dr Sarah Smalley, worked in my lab for 3 months and then left to join a Spin-out company in Cambridge. A Research Assistant followed up the work and we validated these biomarkers. We are now seeking futher funding from the Mfanwy Townsend Trust to finish up and publish this work because the Blond McIndoe has had to close its labs.
Collaborator Contribution Our partners provided the salary for the postdoc plus consumables
Impact We have a formal collaboration agreement together with a MTA to for this collaboration. So far, the collaboration has resulted in three conference presentations. We are at present writing up this work.
Start Year 2012
 
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 2007,2010,2011,2012,2013,2014,2015,2016
 
Description Christmas Stocking Event December 2008 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact The Christmas Stocking Event was a large "speed dating" event to foster collaborations between academics, industrialists and clinicians. A number of new research collaborations resulted from this.

A number of new research collaborations resulted from this activity
Year(s) Of Engagement Activity 2008
URL http://www.sussex.ac.uk/internal/bulletin/archive/07mar08/article7.shtml
 
Description CurioUS Event 2009 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact This was a large event where we had the opportunity to talk to Industrialists/local benefactors about our research. The meeting sparked funding and collaborations for a wide variety of projects.

The meeting sparked funding and collaborations for a wide variety of projects.
Year(s) Of Engagement Activity 2009
 
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-2006,2006,2007,2008,2009,2010,2011,2012,2013,2014,2015,2016
 
Description Visit to Meridian Community Primary School, Peacehaven, 2012. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact The children were interested in genetics and asked lots of questions. They were particularly impressed with Chris Jones' ability to roll his tongue into three folds (rather than the usual one fold)!!

The children asked about entry into University Degrees in Biology. The visit was reported in the BSMS Newsletter (April, 2012).
Year(s) Of Engagement Activity 2012
URL http://www.bsms.ac.uk/about/newsletters