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

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.

This project will decipher the role of the splicing regulator protein Tra2b during the germ cell development pathway that creates sperm. Germ cells are non-essential for viability and the first developmental wave after birth is synchronous. This means we can easily monitor effects of Tra2b deletion on cell types and molecular targets. Preliminary global RNA-binding site data from adult testis predicts that Tra2b regulates genes important for germ cell development. To test this we will use conditional mouse models where we can inactivate production of Tra2b protein in any germ cell type, and monitor effects on cell types using histology and gene expression by RNAseq. Our preliminary data shows Tra2b expression is critical for gonocyte survival, and has identified a small group of splicing targets in genes that control RNA biology and transposable element activity. We will complete identification of target exons controlled by Tra2b in gonocytes, confirm Tra2b binding patterns using gel shifts, and test for effects on encoded proteins and downstream pathways that could be important in gonocyte biology. Tra2b expression increases as cells enter meiosis. In our second aim we will test why this is important by inactivating Tra2b during meiotic prophase. We will use histology to monitor the cellular effects of losing Tra2b on meiosis, and RNAseq to identify the genes, cellular pathways and downstream proteins controlled by Tra2b during meiosis. Our final aim will be to identify the function of a highly conserved splicing feedback control pathway that is conserved in all vertebrates. This feedback pathway is highly active in the testis, and we predict will be important to ensure either stable mRNA splice isoform production during meiosis, or to protect the overall integrity of the transcriptome. We will test this using a new mouse model being made for us by the GEMM programme, analysing the function of this poison exon in meiosis using histology and RNAseq.

BENEFIT TO SOCIETY
WHO WILL BENEFIT. We will contribute to cultural enrichment, by reaching out to local organisations interested in science and schools. We expect male infertility to be of interest to the public and the press.
HOW WILL SOCIETY BENEFIT FROM THIS RESEARCH? We will provide talks to the general public about this project (Café Scientifique, Year 3). We will host sixth form students who are interested in a career in science or medicine, give talks at Science and Engineering weeks at local schools, and issue press releases with papers. We will participate in our IGM flagship event Genetics Matters held every February/March (Years 1-3 of grant).

DELIVERING AND TRAINING HIGHLY SKILLED RESEARCHERS.
WHO WILL BENEFIT? Professional scientists working on this project and students at Newcastle University.
HOW WILL TRAINING BENEFIT FROM THIS RESEARCH? This project will enhance the professional research skills of Caroline Dalgliesh, who is named Researcher on this project, and who will interact with our colleagues in the USA, and local bioinformatics support to analyse RNAseq data sets. A further important training impact of our work will be in science education. Newcastle University is a research-led university, and work in the lab feeds through into taught classes as well as projects carried out by undergraduate and postgraduate students. In the case of lab-based projects, students get the opportunity to become directly involved in research projects. Both the PI and Researcher on this grant are involved in student project supervision. The impact in science education from this grant will be immediate (with undergraduate projects in Years 1-3). As an illustration, our recent BBSRC grant resulted in a paper in Cell Reports that included work from an undergraduate student that was sufficiently important for her to be a co-author. We will similarly engage future students. Caroline will also be involved in training of PhD students including our new BBSRC DTP student.


BENEFIT TO MEN WITH MALE INFERTILITY
WHO WILL BENEFIT? Infertile men with arrested meiosis are frequently seen in clinics, but there is very little that can be done to diagnose or otherwise help them apart from Y chromosome deletion mapping. Although not life threatening, infertility can be psychologically damaging, particularly if it is of unknown origin.
HOW WILL PATIENTS AND CLINICIANS BENEFIT? Although our project is primarily scientific and involves searching for pathways of gene expression that are important for germ cell development in mice, we will also interact with colleagues within our institute who are investigating human male infertility, and our local IVF clinic which is located next door and employs clinicians treating male infertility. These interactions will enable any overlaps in genes and pathways important both in mouse germ cell development and human infertility to be identified. As a result of these interactions, in the long term it is possible that the research in this grant could therefore increase efficiency within the NHS, influence medical practitioners, and be of potential application in the development of diagnostic kits.

BENEFIT TO SCIENTISTS INTERESTED IN DRUG DEVELOPMENT
WHO WILL BENEFIT? Several studies have shown Tra2b to be upregulated in cancer cells suggesting it as a possible future therapeutic target.
HOW WILL STUDY OF TRA2B LEAD TO THIS BENEFIT? Tra2b functions in cancer cells have been globally characterised, and include roles in DNA damage responses that could be important for modulating responses to chemotherapy. However, the normal physiological targets of Tra2b within healthy cells are much less well understood. Yet if Tra2b is to be considered as a possible therapeutic target in cancer it is crucially important to understand these normal physiological functions within healthy animals.