Dissecting ribosome pausing during embryogenesis: from global and single molecule studies to whole embryo phenotypes

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An organism's genes are the blueprint from which proteins are produced, yet in any given single cell, only a subset of all genes is ultimately decoded to give proteins. As such, the identity and abundance of proteins made in a cell determines whether it will become, say a nerve cell in the brain or an epithelial cell in the intestinal lining. This is particularly striking during the development of an organism, where distinct sets of proteins are made at various stages in growth so that different cell types form. Proteins are chains of amino acids which form the building blocks of life. The order and identity of amino acids in a protein is decoded from an intermediate copy of genes called a mRNA in a process termed 'translation'. Ribosomes are the machines inside cells responsible for translation. They bind to the mRNA and sequentially insert amino acids onto a growing protein chain according to the code read from the mRNA. However, not all amino acid insertion events occur with the same efficiency and for many reasons ribosomes pause at sticky patches on a mRNA. In various biological contexts these ribosome pauses are important. However, only a handful of ribosome pausing events have been studied in great detail owing to the technical challenges of following ribosomes on single molecules of mRNA.Our research studies ribosome pausing using the classic model, the fruitfly Drosophila, since the development of an adult fruitfly from a fertilised egg only takes ten days and represents a wonderful system to study how changes in protein production affect the development of a complex multicellular animal. Even at the earliest stages in the fruitfly embryo, the basic segmented pattern of the adult body is becoming apparent. Therefore, the precisely timed production of key proteins at specific sites within the embryo dictates which tissues will develop, and when/ where they will do so. In this proposal, we aim to determine how ribosome pausing controls the timing of protein production so that the correct cell types form during embryonic development. To achieve this goal, we will use state-of-the-art microscopy and sequencing approaches, which will allow us to answer three key questions. Firstly, which mRNAs have paused ribosomes in the fruitfly embryo? Secondly, what is it about these mRNAs that makes the ribosomes pause? Thirdly, how does disrupting ribosome pausing affect development of the fruitfly embryo?Overall, our data will provide important new information about the signals that direct ribosome pausing and the range of processes affected during embryonic development. As all known mechanisms of translational control are conserved across animal and plant cells, results from this study will be directly relevant to human development. Therefore, our findings will be important for understanding the many human diseases that are associated with misregulation of translation elongation. Finally, our data will also benefit stem cell research, where the ability to manipulate gene expression to efficiently differentiate stem cells into particular cell types is a major therapeutic goal.