Functional characterisation of the evolutionarily conserved splicing regulator protein Tra2B in germ cell development

A key question of interest to biologists is how complex organisms like humans and mice can exist with relatively low gene numbers. Embedded within pre-mRNAs are target sites for RNA binding proteins which control nuclear RNA processing so a single gene can encode different mRNAs and protein isoforms. Identifying these binding sites for important nuclear RNA binding proteins, and their functional readout is very important to understand how the genome works. We want to help answer this question using a systems-wide approach to identify functional targets regulated by the nuclear RNA binding protein Tra2B, and to find how physiological splicing patterns are established through Tra2B interactions with hnRNP G family RNA binding proteins. Our results will be significant in understanding how splicing factors interact to enable flexible use of information in the genome and development of complex tissues and specialised cell types. Three aspects of this project are timely. (1) We recently found that important human and mouse transcription factors are controlled by alternative splicing regulation by Tra2B and hnRNP G proteins (BBSRC funded work published in the November 2009 issue of PLoS Genetics and the January 2010 Journal of Cell Science). (2) A conditional version of the gene encoding Tra2B is available for this project and this can be inactivated in any cell type -we will target germ cells. (3) As a first step to comprehensively identify Tra2B-regulated RNAs at a systems-wide level we have identified and mapped 53,000 Tra2B binding sites within the mouse germ cell transcriptome. These sites provide a rich pool of regulated alternative events for analysis in this project. We predict just a subset of these Tra2B binding sites will affect splicing, and a key aim of our grant is to identify these and their functional outcome. Aims and objectives: We will first test whether Tra2B protein is essential for mouse male germ cell development using a conditional mouse strain from which we can remove the gene encoding Tra2B by germ cell-specific coexpression of a DNA cutting enzyme called Cre recombinase. Tra2 is essential for spermatogenesis in fruit flies. We will analyse mice for defects in testis structure or sperm morphology as a consequence of removal of Tra2B, and purify RNA for molecular analysis. We will use this RNA and our transcriptome-wide data to analyse how Tra2B protein regulates alternative mRNA isoforms in mouse germ cells. Our RNA binding data suggests that Tra2B protein binds to a 'poison exon' containing a translational STOP codon in the pre-mRNA for a Tra2B-related protein called Tra2A. We will test if splicing of the Tra2A poison exon changes in the conditional knockout mice. RNA analyses will be done using RT-PCR, with which we can rapidly amplify and characterise different mRNA isoforms from wild type and knockout mice. Because Tra2A might be affected in knockout mice and provide partially redundant function, we will also analyse Tra2B target RNAs by using shortened genes called minigenes that encode pre-mRNAs we can ectopically express in cell lines along with different levels of Tra2B and Tra2A. Since splicing targets respond in a dose-dependent fashion to the concentration of splicing regulators, our prediction is that co-expressed Tra2B will bind to functionally relevant sites and cause splicing affects which we will be able to monitor. We will use these same minigenes to test the splicing effect of protein interactions of Tra2B with hnRNP G family proteins (particularly RBMY and hnRNP G-T: both these proteins interact with Tra2B and are also implicated in controlling normal human fertility). Amongst our current set of Tra2B RNA targets are some which might affect other aspects of mRNA metabolism which we will test to see if Tra2B has more general functions. This project will establish the rules and provide a system-wide picture by which Tra2B protein regulates RNA processing in the male germline.

This project will decipher the role of the important RNA binding protein Tra2B in normal mammalian germ cell development, the extent of functional overlap between Tra2B and Tra2A proteins and the system-wide outcome for splicing control of physical interactions between Tra2B and hnRNP G proteins. We will inactivate an existing conditional allele of the Sfrs10 gene to knock out Tra2B protein in germ cells using germ cell-specific Cre expressed from a VASA promoter. We will analyse effects in the testis using histology of wax embedded tissue, and analyse numbers and quality of any sperm produced. In preliminary experiments to this proposal we have identified 53,000 transcriptome-wide binding sites for Tra2B in mouse germ cells using HITS-CLIP: the most abundant hexamer identified in this screen was GAAGAA which is a known Tra2B binding site. We will use existing Illumina technology to expand our coverage of Tra2B binding sites in the mouse germ cell transcriptome by 10 million new reads. Our current data set predicts a set of alternative splice events under control of Tra2B which we will test by RT-PCR analysis in vivo in mice and in vitro using minigenes . We will correlate the positions of Tra2B binding sites with the outcome of splicing regulation. Tra2A is a particularly strong candidate for Tra2B regulation, as is the NASP pre-mRNA encoding a chromatin modifying protein, and both alternative splices are evolutionarily very conserved. We will use minigenes to analyse at a systems-wide level how Tra2B target RNAs are co-regulated through interactions with the hnRNP G family proteins RBMY and hnRNP G-T, and integrate our transcriptome map of Tra2B binding sites with existing transcriptome-wide RNA target sites identified for hnRNP G-T to identify target RNAs bound and coregulated by both proteins. We will also analyse potential novel functions of Tra2B outside splicing control, particularly in 3' end formation.

A major social and economic impact of our study will be in the continued development of the local science infrastructure. Science is a central theme of the local Regional Development Agency, One North East, set up in 1999 to sustain jobs and prosperity in the North East of England and funded by Central Government. Newcastle is a post-industrial city in which the University is a major local employer, and is developing itself as a Science City in which the economy is based on a foundation of research and technology. Within the North East of England over 140,000 people are employed in biotechnology, life sciences, NHS and associated organisations. The Institute for Human Genetics (IHG) is part of the city centre-located International Centre for Life, which includes both aspects of Science education and start up companies, and links with local patient care under the NHS. Key to this vision is Research Excellence. The creation of an excellent basic research base has acted as a magnet to attract both high technology companies and further local investment in healthcare. The excellent reputation of Newcastle University and other local universities in both basic and applied scientific research is critical for the success of Newcastle as a Science city, and continued Research Council funding of excellent science is crucial for this. Our current project involves international collaboration with scientists in Cologne, and builds on the status of Newcastle as an existing international scientific hub to which we have also contributed by our establishment of a Royal Society link with the IGBMC in Strasbourg. In this project we will use expertise from a post-genomic startup company we have successfully worked with in Paris, thus assisting the development of Newcastle as part of the EU network of interacting scientists and technologists. A major impact of this project will be to contribute to local scientific training within Newcastle. This project will train an existing member of my group in new genetic techniques. Expanding expertise in the application of post-genomic technology to basic research such as we plan in this project can make valuable contributions industrially. As an example of this, the June 2007 issue of The Economist magazine had a front cover dedicated to RNA, which it described as Biology's Big Bang. Although our basic research is 'blue skies' in nature, it does have significant translational impacts in normal health and medicine and we have collaborated with clinicians and other research groups to realise these. We have also trained a Clinical Training Fellow within the group to PhD level, who is now a urological surgeon doing translational research. We also expect our project to have impact in terms of public engagement. We are situated next door to a Life Science museum and this provides a natural outlet for the public engagement activities of myself and several members of the research team. The activities, in particular the regular 'meet the scientist' event initiated by a postdoctoral Fellow in my group, has led to others from the IHG getting involved.