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

点击翻译按钮获取中文摘要

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.