Analysis of cis-acting RNA sequences required for intracellular localisation of gurken,the Drosophila TGF alpha homologu

Lead Research Organisation: University of Oxford
Department Name: Biochemistry

Abstract

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Technical Summary

Intracellular mRNA localisation targets proteins to their cytoplasmic site of function and is important in many symmetry breaking processes ranging from yeast mating type switching and actin based cell motility, to embryonic axis specification in Drosophila and Xenopus. We have been studying the mechanism of asymmetric localisation of Drosophila gurken (grk), a TGFa homologue that plays a central role in the specification of the major body axes. grk mRNA is localised to one side of the oocyte nucleus, restricting the distribution of the Grk signal that leads to the establishment of the antero-posterior and the dorso-ventral axes of the oocyte and the embryo. The mechanism of grk mRNA localisation remains poorly understood largely because of the lack of rapid in vivo and in vitro assays for localisation. We have developed a novel and rapid in vivo assay by injecting fluorescently labeled grk RNA and imaging its assembly into particles that localise correctly in living oocytes and embryos (Appendix I). The assay will allow us to determine the mechanism and route of localisation. We will also define systematically the cis-acting sequences and trans-acting factors necessary and sufficient for grk mRNA particle formation and localisation. We have also been developing an in vitro assay for binding of grk RNA to microtubules and motility to their minus ends, using ovary or embryonic extracts added to fluorescent RNA and in vitro assembled microtubule asters (Appendix II). We will use the assay to demonstrate and study microtubule based motility and purify trans-acting factors that bind to specific cis-acting elements. In June 2000, I began a 5 year Lister Senior Fellowship.

Publications

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Beckett MG (2009) Building a scientific data grid with DiGS. in Philosophical transactions. Series A, Mathematical, physical, and engineering sciences

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Hamilton RS (2007) RNA localization signals: deciphering the message with bioinformatics. in Seminars in cell & developmental biology

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Hamilton RS (2011) Identifying and searching for conserved RNA localisation signals. in Methods in molecular biology (Clifton, N.J.)

 
Description Wellcome Trust strategic review of funding policy for advance microscopy infrastructure
Geographic Reach National 
Policy Influence Type Participation in advisory committee
Impact Led to the Wellcome Trust funding more microscopy development and infrastructure
 
Description BBSRC funding (Role of mRNA localization in transposable element biology)
Amount £250,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2007 
End 08/2010
 
Description EP Abraham Cephalosporin Trust Fund
Amount £85,000 (GBP)
Organisation University of Oxford 
Department E P A Cephalosporin Fund
Sector Charity/Non Profit
Country United Kingdom
Start 02/2007 
End 01/2010
 
Description EPSRC funding EP/F019165/1 A spatio-temporally integrated and nonlinear particle tracking system for live cell imaging
Amount £269,120 (GBP)
Funding ID EP/F019165/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 02/2008 
End 01/2011
 
Description Fell fund
Amount £70,000 (GBP)
Organisation University of Oxford 
Department John Fell Fund
Sector Academic/University
Country United Kingdom
Start 09/2007 
End 08/2009
 
Description Wavefront metrology
Amount £17,610 (GBP)
Organisation Science and Technologies Facilities Council (STFC) 
Sector Academic/University
Country United Kingdom
Start 02/2007 
End 01/2010
 
Description Wellcome Trust Senior Res. F'Ships In Basic Biomedical Science (Elucidating the molecular mechanisms of intracellular mRNA transport and anchoring)
Amount £2,046,000 (GBP)
Funding ID 081858/Z/06/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2007 
End 02/2012
 
Description Wellcome Trust Senior Res. F'Ships In Basic Biomedical Science (Investigation of the machinery required for mRNA localisation in Drosophila)
Amount £1,507,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2003 
End 06/2007
 
Description Wellcome Trust Strategic award
Amount £3,900,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2011 
End 07/2016
 
Description A spatio-temporally integrated and nonlinear particle gtracking system for live cell imaging. 
Organisation Heriot-Watt University
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided the live cell imaging data and Weiping and his team provided novel algorithms for denoising and particle tracking for us to test and improve.
Collaborator Contribution We have together developed new algorithms for denoising and tracking EB1 MT growth and polarity data. This will be published this year.
Impact These findings will be published as new algorithms in Engineering/Physics journals and the biological data will be published in biological journals.
Start Year 2008
 
Description In vivo proteomics in Drosophila 
Organisation University of Cambridge
Department Gurdon Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution A gene trap screen
Collaborator Contribution We were part of a large collaborative screen coordinated by St Johnston in Cambridge. We screened the collection for macrophage expression and found some interesting new patterns. St Johnston mapped all the inserts and screened them for oocyte expression patterns.
Impact No publications yet, but the screen found many very interesting new genes and expression patterns.
 
Description The role of mRNA localization in transposable element biology 
Organisation University of Edinburgh
Department Institute of Cell Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided the know how for in vivo mRNA localization injection assays and advanced microsocpy.
Collaborator Contribution Prof. David Finnegan's group provided the know how for biological analysis of transposable elements and we provided the know how for microsinjection assays and advanced imaging in Drosophila oocytes and embryos.
Impact 19144907 15992540
 
Description High school children. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact I gave a presentation to High school children as part of a regular public engagement excercise to help them decide which science they should choose to study at University.

High school children across Scotland were aided in their effort to make appropriate choices for University courses and were enriched in their general understanding of Biology.
Year(s) Of Engagement Activity 2007
 
Description Primary school teacher microscopy training 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Primary Audience Schools
Results and Impact I help equip St Nicholas primary school in Oxford with microscopes, and taught all the interested shcool teachers how to operate the microscopes and teach school children their use.

The primary school children at the school were and will be exposed to microscopy and some basic observation of micro-organisms.
Year(s) Of Engagement Activity 2008
 
Description Scottich High School Head teachers / Heads of Biology 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Primary Audience Schools
Results and Impact Approximately 20 heads of Biology or heads of schools from across Scotland attended the course annually. They were exposed to basic molecular biology methods such as PCR and restriction enzyme digests as well as to some short presentations from researchers.

This was greatly appreciated by the teachers, as they were exposed for the first time to the instruments and techniques they taught about theoretically. They returned to their schools with hugely improved enthusiasm and understanding of modern Biology.
Year(s) Of Engagement Activity 2006