Investigating novel steps for promoting tRNA binding to translation factor eIF2 during protein synthesis initiation

All organisms are composed of cells. Cell growth and cell division are coordinated and controlled by a wide range of signals that ensure they occur only at appropriate times. At the extremes, developing embryos require rapid growth while adults require much slower growth to replace damaged or dying cells. When there is a loss of growth control, diseases such as cancer can develop, while a failure to promote growth when required can cause a failure to repair damaged cells or cause tissue wasting. We have been studying how cells control the conversion of nutrients into the new proteins that are required for life. Almost all cellular functions are performed by proteins. Each one is made from building blocks called amino acids that are linked in chains and folded to make 3-dimensional structures that are important for each to fulfil their individual roles. The instructions required to make each protein correctly are determined by the DNA sequences of the genes in the genome. This is termed 'protein synthesis' and it is the final step in the pathway called 'gene expression' which is critical for ensuring that the correct genes are decoded at the correct place and time.
Protein synthesis occurs within molecular machines called ribosomes that decode instructions relayed from the genome within intermediary molecules called messenger RNAs (mRNAs). Human cells each contain over a million ribosomes. mRNA decoding by ribosomes is made possible by the concerted action of 'helpers' called protein synthesis factors and adapter molecules called transfer RNAs (tRNAs) that bring the necessary amino acids together. This proposal concerns the initiation phase in which a dedicated set of factors act.. Protein synthesis initiation factors direct the ribosome and a specialised tRNA called initiator tRNA that starts proteins with the amino acid methionine (designated Met-tRNAi) to the correct start place on each mRNA. This is critical to make the right proteins in every cell. This must be done both accurately and rapidly Initiation is the most complex phase of protein synthesis and the least well understood at the molecular level.. This proposal concerns factors designated eIF2B, eIF2 and Met-tRNAi.
In this proposal we describe preliminary experiments that have uncovered a novel function for the factor eIF2B.
eIF2B is known as a factor that 'switches on' its partner eIF2 so that eIF2 can bind to Met-tRNAi and recruit it to ribosomes. We have found that eIF2B has a second role to remove another factor (eIF5) from eIF2. This happens before eIF2B switches on eIF2. eIF2B is a complicated protein with five parts and both the existing and new roles only require 2 of them. Genetic and biochemical studies, including many done in our laboratory, indicate that eIF2B has further essential roles and here we outline our plan to investigate those. Specifically we will evaluate how eIF2B accelerates Met-tRNAi binding to eIF2 and how Met-tRNAi then promotes removal of eIF2B. As eIF2B mutations cause the fatal brain disease called 'Vanishing white matter disease' (VWM) we will investigate how the mutations causing VWM alter these new activities. Because protein synthesis is one of several functions critical for all our cells we believe that it is important to study this now, while we have a lead in this area of research.
By providing a detailed understanding of the contribution of eIF2B to the control of protein synthesis it will help understand control of cell growth and provide further insight into how VWM causes disease. The work may also be of interest to industries eg those that produce specific proteins as drug therapeutics or for commercial products or those that grow cells by fermentation because it will allow an improved understanding of protein synthesis mechanism. By understanding the precise controls of protein synthesis this may help in the design optimized commercial protein expression or fermentation systems.

Unravelling the nature and control of molecular interactions drives much molecular biological research. For example the large G protein family function as molecular switches, controlled by interacting ligands and regulatory partners. In many G protein systems studied, switch specificity is governed by restricted subcellular localization of regulators that control the GTP/GDP bound forms of their cognate G protein. However, in protein synthesis neither sub-cellular localization or nucleotide status appears sufficient to explain the efficient control of the translation factor G protein eIF2 by protein ligands. In protein synthesis eIF2 functions to bind initiator tRNA (Met-tRNAi) to ribosomes in all eukaryotic cells. Its GTP/GDP status is controlled by two factors, eIF2B (GEF) and eIF5 (GAP). We showed that eIF5 has a second function preventing GDP release from eIF2/GDP (GDI). Several observations are important for this proposal. 1) We find that eIF2B has a second function as a GDI displacement factor (GDF). 2) The GDF and GEF functions of eIF2B require only 2 of the 4 essential subunits of eIF2B, raising questions as to what the essential functions of the other two subunits are? 3) The binding affinity of eIF2B and eIF5 for eIF2 is not altered by GTP/GDP status, so that they alone cannot drive forward momentum of the translation cycle. It appears likely that Met-tRNAi binding to eIF2 does this. In the case for support we outline several lines of preliminary data which support the hypothesis that eIF2B has additional functions necessary to promote Met-tRNAi binding to eIF2 and release eIF2B to facilitate efficient protein synthesis initiation. We propose a series of biochemical experiments to test this idea using highly purified reagents available in our laboratory. We propose supporting molecular studies to define which eIF2B elements are critical for its functions. Together these studies will biochemically define new steps in the global protein synthesis pathway.

This work will examine the mechanisms of regulation of translation in eukaryotes by the protein synthesis factor eIF2B. Yeast has served as a paradigm for the study of translational regulation across all eukaryotes. Much of the current understanding in the translation control field has stemmed from this work in yeast. Outside of the immediate academic fields the research may be of interest to clinical scientists, medical practitioners, patient groups, industrial scientists and the wider public. Scientists/ clinicians are seeking to control protein synthesis therapeutically. For example, developing specific inhibitors for use in cancer treatments. In addition mutations in eIF2B cause a fatal human disorder-VWM. This has raised interest in translational control and in my laboratory's research studies. The disease-causing mutations affect the ability of glial cells to correctly form myelin sheaths around neuronal axons, and manifests as ataxia. This is a chronic-progressive disorder; patients suffer seizures, comas and deteriorate leading to a premature death. Our results may impact on the understanding of VWM.
To promote understanding of research findings and communication, GP joined the management committee of an EU-funded COST (Co-operation in Science and Technology) Action called MYELINET (Myelin Orphan Diseases in Health), formed to promote understanding and to coordinate the study, training and treatment for the many orphan diseases affecting the CNS myelin. The European Leukodystrophy Association (ELA) represents patient groups and organise patient-scientist meetings to facilitate face-to-face contact between researchers and to communicate their research findings directly to patients/ parents. GP spoke at a meeting in 2009. Myelinet has been superseded by an EU-FP7 program grant to which GP contributes. GP has also presented our work to clinical geneticists at Manchester children's hospital (Feb 2012). GP will continue to pursue/engage with these clinical/ patient groups to promote greater awareness and understanding of our research to promote its impact.
The project may have industrial interest. Bulk growth of yeast is used to produce a variety organic molecules, beverages/food stuffs and biopharmaceuticals. Applications include where there is a requirement to highly express specific proteins, for example therapeutic or industrially relevant proteins. Translational control is clearly linked to both bulk cellular growth and recombinant protein expression, therefore a greater understanding of these processes can only benefit industries using such methods. The Centre of Excellence for Biopharmaceuticals (COEBP) within FLS at Manchester actively engages with potential industrial partners. GP and other academics have recently met with several company representatives with the aim of developing commercially relevant projects. Further meetings are planned for 2013. GP will endeavour to make contact with industrial partners to promote our science. Should specific commercial exploitation opportunities arise, we will make full use of the University of Manchester Intellectual Property company (UMIP) to maximize the commercial impact. UMIP has resources and experience to facilitate potential commercial application through contracts, patents and has funding available to pump-prime projects requiring development.
Public engagement is also part of our impact plan. FLS has an annual community open day. GP contributed in 2012 offering the public insight into our science through interactive table-top activities and poster information to engage children and adults alike to promote scientific understanding and enthusiasm. We will run similar activities at future events and expect the employed RA to contribute. We have hosted secondary school work experience students to gain hands on more in-depth experience of lab-life and intend offering similar future places.