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

Reference	BB/X007294/1
Principal Investigator / Supervisor	Professor Hilary Ashe
Co-Investigators / Co-Supervisors	Professor Mark Peter Ashe, Professor Sam Griffiths-Jones
Institution	The University of Manchester
Department	School of Biological Sciences
Funding type	Research
Value (£)	832,799
Status	Current
Type	Research Grant
Start date	01/04/2023
End date	31/03/2026
Duration	36 months

Abstract

The correct regulation of gene expression is fundamental to multicellular life. Translational regulation is widely used to modulate gene expression outputs, with mutations in components of the translation machinery associated with a variety of human diseases. Translation elongation is not uniform, but instead represents a key regulated step that determines translation efficiency. For example, ribosome pausing has recently been described as an important elongation checkpoint. In this proposal, we will therefore test the hypothesis that translation elongation is modulated to control developmental gene expression and patterning. As such, the global aim of this proposal is to exploit advances in genomics and single molecule live imaging to determine how ribosome pausing regulates patterning in the Drosophila embryo. We will use disome-seq to map the positions of the collided ribosomes (disomes) caused by pausing across the early embryonic transcriptome. Computational approaches will be used to identify mRNA and nascent protein features that direct pausing. These elements will be validated by biochemical analysis using embryonic extracts and by using cutting-edge single molecule live imaging of mRNA translation in the embryo. We will then exploit CRISPR genome editing to mutate ribosome pause sites for a subset of mRNAs, in order to determine the effect on translation, and the precision and robustness of embryonic development. The Drosophila embryo is the ideal model for this study due to its rapid development, ease of manipulation and genome editing, suitability for single molecule live imaging, and the extensive characterisation of the gene regulatory networks underpinning early embryogenesis. Overall, results from this study will not only answer key questions relating to how ribosome pausing regulates developmental gene expression, but our findings will have wider implications for understanding human diseases associated with variant translation elongation factors.

Summary

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.

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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