Identifying the functions of a family of nuclear RNA binding proteins that switch expression between somatic and meiotic cells

PURPOSE OF RESEARCH. Vertebrate genes make extensively processed RNAs guided by RNA binding proteins that are often encoded by multigene families. However, the individual functions of proteins within these families are frequently not well understood. The purpose of this research is to provide the first global analysis of the RBMX gene family which is conserved in all mammals, and has members conserved in all vertebrates. The RBMX family of nuclear RNA binding proteins consists of RBMX, hnRNP GT and RBMY proteins, and is interesting since there are gene expression switches between somatic cells (found all over the body) and meiotic cells (that eventually give rise to sperm). Preliminary mouse knockout data indicates that these gene expression switches are critical, but why this is or what individual RBMX family proteins do is still largely unknown.

TIMELINESS AND VALUE FOR MONEY. We have already established conditions in which we can deplete the RBMX protein in somatic cells, and induce changes in RNA processing of one endogenous target transcript, which we will be able to scale up to a more global analysis. We have also made a new mouse model, from which we can delete the highly conserved Hnrnpgt gene. Although these knockout mice are otherwise healthy, Hnrnpgt deletion causes germ cells to arrest during meiosis at a crucial stage called diplotene. The combination of these two advances, RBMX knockdown and hnRNP GT deletion, provide us the opportunity to understand how the coordination of RBMX and hnRNP GT protein activity controls pathways of nuclear gene expression.

OBJECTIVES. Objective 1 will identify target genes and pathways controlled by the RBMX protein in somatic cells, and mechanisms of RBMX function. Objective 2 will search for genes and pathways regulated by hnRNP GT protein in meiosis, and seek to determine if these are the same or different to those regulated by RBMX in somatic cells. Although we don't know what pathways are disrupted without hnRNP GT protein, we can make a strong prediction from the detailed phenotype of our Hnrnpgt null mice. HnRNP GT null germ cells arrest during meiosis at diplotene, an important stage where the paired chromosomes are just starting to separate. Our preliminary data show that meiotic chromosomes are abnormally spread out in Hnrnpgt null testes, suggesting at least some of the targets of hnRNP GT will control meiotic chromosome structure. Interestingly RBMX has also been connected to chromosome structure, but the mechanism is not known. Objective 3 will take further advantage of our new mouse model to determine if hnRNP GT has important functions after meiosis, during which time RBMX is still turned off. Objective 4 will test if the RNA processing pathways we find to be controlled by mouse RBMX and hnRNP GT are conserved in humans, and mis-regulated in infertile men.

Taken together, these objectives will dissect individual functions within this highly conserved family of RNA binding proteins, and how switches in expression between individual proteins control global patterns of gene expression. In particular, we will test how the switch between RBMX and hnRNP GT expression contributes to changes in global splicing programmes.

OUTCOMES. The expected outcomes from this research will be a better understanding of how families of nuclear RNA binding proteins function to control pathways of development. Such programmes are likely important all over the body. This project will shed new light on how the RBMX proteins are important to human health. RBMX family genes are implicated in human male infertility, brain and muscle development and cancer, but still poorly understood. This would be the first comprehensive analysis of this family of RNA binding proteins. The beneficiaries of this project will be scientists interested in gene expression and development, scientists and students that we train, infertile men and members of the public with whom we engage.

This project will address functions within a protein family of nuclear RNA binding proteins that switch expression between somatic body cells (most of the cells in the body) and meiotically active cells (that ultimately make the next generation).

We will decipher global mechanisms of RNA processing control by RBMX using already established protocols to deplete RBMX expression, and then RNAseq to identify direct and indirect targets. Direct RBMX targets will be distinguished using iCLIP (a powerful crosslinking approach that can reveal global endogenous RNA targets that are bound to RBMX). RBMX protein turns off in meiosis and is replaced by another family member called hnRNP GT. In our preliminary experiments we find this switch in gene expression is crucial, since mice null for the Hnrnpgt gene arrest germ cell development during meiosis at a stage called diplotene. Both wild type and Hnrnpgt null mice have similar cell type contents at developmental stages before this block (postnatal day 18). We will globally identify RNA targets of hnRNP GT protein by RNAseq, comparing day 18 mouse testes from wild type and knockout mice to identify mis-regulated RNA processing pathways. Direct hnRNP GT target RNAs will be identified using iCLIP to address patterns and mechanisms of regulation, and to what extent these are similar or overlap with RBMX. We will monitor RNA processing pathways of endogenous transcripts over germ cell development to establish whether hnRNP GT provides a direct or specialised replacement for RBMX in meiosis, and how this fits into the global transitions in RNA processing pathways occuring during germ cell development. To establish if hnRNP GT is also needed after meiosis, we will cross our floxxed mouse model with a Prm-Cre expressing mouse to inactivate hnRNP GT after diplotene. Finally we will examine RNA processing patterns in human testis RNA to see if the RNA processing events controlled by RBMX and hnRNP GT are conserved.

BENEFIT TO PATIENTS WITH MALE INFERTILITY AND THEIR CLINICIANS
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.
HOW WILL PATIENTS AND CLINICIANS BENEFIT? The identification of new pathways important for completion of meiosis will potentially lead to new genetic tests, and in the long term to the development of blood-based tests that could obviate the need for testis biopsies that are expensive and potentially hazardous. 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. We will also interact with the Infertility Network UK, a charity that promotes the interests of people with infertility. Although not life threatening, infertility can be psychologically damaging, particularly if it is of unknown origin.

DELIVERING AND TRAINING HIGHLY SKILLED RESEARCHERS.
WHO WILL BENEFIT? Students at Newcastle University and professional scientists working on the project.
HOW WILL TRAINING BENEFIT FROM THIS RESEARCH? An important 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 for a time in research projects, and both the PI and Research Coinvestigator on this grant are involved in project student supervision. The impact in science education from this grant will be immediate (Years 1-3).This project will also enhance the professional research skills of the Research Co-investigator on this project, who will interact with local bioinformatics support for the RNAseq analysis.

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 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 (Years 1-3 of grant).


ENHANCING THE KNOWLEDGE ECONOMY
WHO WILL BENEFIT? A longer term social and economic impact of our study will be in the continued development of the local science infrastructure. 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 of Genetic Medicine (IGM) 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.
HOW WILL THE LOCAL ECONOMY BENEFIT FROM THIS RESEARCH? Local expertise in the application of post genomic technologies will make an important contribution to the local scientific infrastructure. Our high profile research also acts to bring in international students and scientists, which has a positive knock on effect on the local economy.