Award details

The re-modelling of mRNPs and the regulation of localised mRNA translation during mammalian cell attachment and spreading

Reference	BB/L018209/1
Principal Investigator / Supervisor	Professor Simon Morley
Co-Investigators / Co-Supervisors
Institution	University of Sussex
Department	Sch of Life Sciences
Funding type	Research
Value (£)	331,311
Status	Completed
Type	Research Grant
Start date	30/11/2014
End date	29/11/2017
Duration	36 months

Abstract

Protein synthesis is carried out in three linked stages (initiation, elongation and termination), with binding of mRNA to the ribosome a major site of translational regulation. This step is facilitated by the assembly of initiation factors (eIFs) into a multi-protein complex, eIF4F (eIF4E, eIF4A, eIF4G), associated with eIF3 and the ribosome. eIF4F formation is strictly controlled by both regulatory proteins (such as 4E-BP1 and CYFIP) and their intracellular localisation, regulating cell migration, growth and survival. Localised protein synthesis coupled to mRNA targeting spatially restricts the synthesis of specific proteins required for cell spreading and migration; how the cell regulates this is unclear. With MRC5 fibroblasts, we have used confocal microscopy and IF to show an enrichment of initiation factors, actively translating ribosomes, specific mRNAs and novel regulatory mRNA binding proteins (CYFIP, FMRP) in lamellipodia at the leading edges of spreading cells. Furthermore, FISH, IP and TIRF microscopy show that mRNAs encoding structural and regulatory proteins of the WAVE complex are in the same mRNP complexes as FMRP/CYFIP at the leading edge. As protein synthesis is a requirement for cell spreading, we suggest that the regulation of localised translation has a pivotal role in controlling cell spreading and migration. However, outside of neuronal cells, little is known about how FMRP/CYFIP regulate localised translation during cell spreading. Loss of CYFIP, a negative regulator of eIF4E, is associated with increased mobility on tumour cells. To address this, we will use fibroblasts and: (a) determine if there is a regulatory role for phosphorylation and/or arginine methylation of FMRP during cell spreading; (b) analyse whether CYFIP/FMRP/eIF4E complexes are regulated at the leading edge of cells; (c) investigate structural elements in target mRNAs required for recruiting such mRNA to regulatory binding proteins in lamellopodia.

Summary

During cell migration, critical information stored in the genetic material (DNA) has to be decoded by the cell to produce a wide variety of proteins in the right amount, place and time to allow for this process occur; tumour cells often get this wrong! The general transfer of information from DNA to protein is carried out by the messenger RNA (mRNA), which is a copy of the DNA sequence. This mRNA has to be decoded into protein by a complex, highly regulated machine termed a ribosome, in a process known as translation. To work efficiently, accurately, and to allow the ribosome to function in the best interests of the cell, this machinery requires helper proteins (translation initiation factors; eIF) that interact with each other, and also make sure that the mRNA and the ribosome come together into a highly regulated, large initiation complex to make the proteins required. So how does the cell control this? The interaction of the initiation factors themselves is a major site for regulation in mammalian cells. Regulatory proteins, such as 4E-BP1 and CYFIP, prevent the interaction of eIF4E with the scaffold protein, eIF4G, and stop the recruitment of mRNA to the ribosome and halt protein synthesis. When protein synthesis is needed, the cell signals for the release the eIF4E/mRNA from the inhibited complex to let it work when and where it is needed. However, we still do not know how the cell controls exactly where protein synthesis is activated in cells which are in the process of spreading and migrating. From "looking" inside the cell with specialised microscopy techniques, we know that the initiation factors and their regulatory proteins are discretely localised to the edge of cells in the direction that they are moving; they are not just floating about. In the work described here we want to investigate the nature of the specialised signals used by the cell to regulate localised protein synthesis, look at the complexes of proteins and mRNA found at such sitesand understand what parts of the mRNA make it attractive to these mRNA binding proteins at the edge of cells. These studies will substantially increase our general understanding of the significance of the control of protein synthesis in the regulation of cell growth and migration, opening up new potential avenues for controlling cancer cells which have acquired the ability to move about the body.

Impact Summary

In terms of the impact of our proposed work, the main beneficiaries of our basic, blue-sky research will be academic colleagues and those in the Pharma industry with an interest in cell migration, growth control and survival. The work described here is a novel avenue of research which will investigate the importance of phosphorylation and arginine methylation on protein complex assembly and selective mRNA binding at the leading edge of spreading fibroblasts. In terms of IP exploitation and technology transfer activity, Sussex has mature mechanisms to evaluate, exploit and protect IP arising from research projects. Another avenue for impact will be the successful application for patents. In addition, we have set up new, cross-field collaborations for promoting this research and training opportunities for staff, helping us to maximize the impact of this publicly funded research. Over many years, significant progress has been made in identifying protein complexes which regulate localised protein synthesis in neuronal cells. However, little is known about the role of such complexes and their modification by phosphorylation and/or arginine methylation in regulating selective mRNA translation in spreading fibroblasts. This particular proposal adopts a multidisciplinary approach to understanding the role for phosphorylation and/or arginine methylation in mRNA/protein complex assembly and activity, opening up a whole new avenue of research in the UK. This will also promote the career of the PDRA by allowing him/her to master our puromycylation assay, biochemical analysis of protein complexes, and the use of scanning confocal microscopy and TIRF microscopy for IF and FISH analysis of proteins and mRNA, respectively. In addition the PDRA will learn to identify novel sites of protein modification using in-house mass spectrometry. This study will not only improve our understanding of the importance of translational control in regulating cell growth, proliferation,survival and migration, but will also provide valuable insight into the specific control of localised translational machinery and how this process might be targeted by the development of novel therapeutics.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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