Why is the highly conserved splicing regulator protein Tra2b essential for spermatogenesis?

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PURPOSE OF RESEARCH: RNA binding proteins like Tra2b play a key role in organising gene expression. This includes the process of alternative splicing through which most single genes produce multiple mRNA splice isoforms. Alternative splicing plays a critical role in building complex animal bodies. Since Tra2b is required for whole animal viability in mice the global targets and pathways it regulates in healthy animals are much less understood compared to its role in cancer cells. The purpose of this research is to address this knowledge gap in the genetically tractable germ cell developmental pathway that makes sperm, and involves cell division by mitosis and meiosis and cell differentiation.TIMELINESS AND VALUE FOR MONEY: Four aspects of this project are particularly timely. 1) We have an existing conditional Tra2b knockout mouse that we can inactivate within germ cells (in the developmental pathway leading to sperm). Germ cell development is highly active in adult mice yet not essential for viability. Our preliminary work shows that Tra2b is critical for the survival of an important group of germ cells called gonocytes (these are precursors to all other downstream germ cell developmental stages). 2) Through international collaboration with a world leading group in the USA we will be able to identify Tra2b splicing targets in genetically modified germ cells. This collaboration has already enabled us to identify initial splicing targets for Tra2b within gonocytes, and will also help us later in the project. 3) We have identified several million RNA-binding sites for Tra2b using cross linking and next generation sequencing. 4) The MRC unit at Harwell are making a mouse model that will enable us to probe the function of a splicing feedback pathway that controls expression levels of Tra2b and is up-regulated in the testis. This model will later be generally available, but there is a window of opportunity where we will have sole access and can make a really important contribution to understanding why this feedback pathway is important.AIMS AND OBJECTIVES: This project will decipher how the splicing activity of Tra2b controls germ cell development. Our first objective will be to identify splicing targets controlled by Tra2b in gonocytes, and to analyse these to identify gene expression pathways important for gonocyte survival. Our second objective will be to identify targets for Tra2b during meiosis. We want to do this since Tra2b is up-regulated at meiosis. Our RNA-binding data predicts that Tra2b controls important meiotic genes, and predicts mechanisms of Tra2b splicing control that we want to globally test. Our third objective is to test how Tra2b splicing activity during meiosis is controlled by a splicing feedback control pathway. This splicing feedback control involves an ultraconserved region of the genome indicating it is very important, and is very active in meiotic cells, yet it has not been functionally investigated within an animal model. We predict that this feedback control pathway might either stabilise normal patterns of meiotic splicing control by Tra2b, or alternatively might prevent Tra2b levels increasing to a point where they can damage the transcriptome.OUTCOMES: We expect that the results of this project will be significant in understanding how splicing factors function to enable flexible use of information in the genome, the development of complex tissues like the testis, and specialised cell types like those undergoing meiosis. Our project will discover new regulated targets of splicing control, and interrogate mechanisms of regulation and phenotypic consequences when this is blocked. The main beneficiaries from this work will be scientists interested in gene expression, scientists and students who will be trained and members of the public that we will engage.