Characterisation of distinct eIF4E mRNA cap binding proteins during early Drosophila development

Cellular growth is fundamentally linked to the levels of proteins synthesised. In order to double in size during cell division, cells must double their protein content. Proteins are made by copying information contained in genes via an intermediate molecule called messenger RNA (mRNA). The process of mRNA production is called transcription, whereas the process by which proteins are made from mRNA is called translation. These processes involve many factors and in the proposal we will concentrate on a specific translation factor, eIF4E, that recognises mRNA. Interestingly many organisms contain a number of different forms of eIF4E that may vary in their role. Both transcription and translation can be controlled, allowing cells to precisely vary which genes are translated into protein. This control is particularly important in multicellular organisms where many distinct types of cell produce different types of protein. The translation factors which regulate translation in simple organisms, such as the fruitfly, are the same as those in humans. As the fruitfly has many advantages as an experimental organism, it is commonly used in research. In the fruitfly embryo, it has been established that translation regulation is critical to its correct development. In this proposal, we will use the fruitfly embryo as a model to investigate if the different forms of the eIF4E translation factor have distinct functions, as well as determining how they are involved in the early stages of embryonic development. Findings from this study will provide insight into potential functions for different eIF4Es in other organisms, including humans where multiple eIF4Es exist.

The central dogma of molecular biology is that DNA is transcribed into mRNA which is then translated into protein. The translation of proteins from mRNA is therefore a fundamental process in all living cells and accounts for a large proportion of cellular activity. The eukaryotic translation initiation factor, eIF4E, is a key regulated component of the translation machinery that selects and directs mRNA for translation. Interestingly in a number of higher eukaryotic species there are several eIF4E genes encoding multiple eIF4E isoforms, although the significance of having more than one eIF4E is poorly understood. In the fruitfly, Drosophila melanogaster, there are seven distinct eIF4E genes. This represents the highest number of eIF4E genes per genome that is currently known. Studies in the Drosophila early embryo have served as a paradigm for the developmental control of gene expression at the level of mRNA localisation and translation. Therefore, the experiments in this proposal aim to use the Drosophila early embryo as a model system to investigate whether the different eIF4E isoforms have distinct functions in development. We show that four of the Drosophila eIF4E genes are expressed at early embryonic stages. Biochemical interaction studies will be combined with genetic approaches in the embryo to elucidate the differential properties of these four eIF4E isoforms. For instance, we will analyse which mRNAs each isoform interacts with in vivo using a defined microarray-based strategy. The analysis of mRNAs that interact with a particular eIF4E protein will be closely correlated with the phenotypes of loss-of-function mutants in each eIF4E gene. The translational function and potential mRNA export functions of each eIF4E protein will be assessed using various complementation strategies and export assays. The overall combined experimental schedule will allow us to understand the developmental and biochemical distinction between different isoforms of this pivotal translation initiation factor.