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

研究目的。脊椎动物的基因在RNA结合蛋白的指导下进行广泛的加工，RNA结合蛋白通常由多基因家族编码。然而，这些家族中蛋白质的个体功能往往没有被很好地理解。本研究的目的是首次对RBMX基因家族进行全球分析，该基因家族在所有哺乳动物中都是保守的，其成员在所有脊椎动物中都是保守的。RBMX家族的核RNA结合蛋白由RBMX、hnRNP GT和RBMY蛋白组成，由于在体细胞(遍布全身)和减数分裂细胞(最终形成精子)之间存在基因表达开关，因此很有趣。初步的小鼠基因敲除数据表明，这些基因表达开关是至关重要的，但为什么会这样，或者单个RBMX家族蛋白的作用在很大程度上仍是未知的。我们已经建立了条件，在这种条件下，我们可以耗尽体细胞中的RBMX蛋白，并诱导一个内源性目标转录本的RNA加工变化，我们将能够扩大到更全局的分析。我们还制作了一个新的小鼠模型，从中我们可以删除高度保守的Hnrnpgt基因。尽管这些基因敲除的小鼠在其他方面都是健康的，但Hnrnpgt缺失会导致生殖细胞在减数分裂的关键阶段双线期停止。RBMX基因敲除和hnRNP GT缺失这两个进展的结合，使我们有机会了解RBMX和hnRNP GT蛋白活性的协调如何调控核基因表达的途径。目的1鉴定RBMX蛋白在体细胞中调控的靶基因和途径，以及RBMX功能的机制。目的2寻找在减数分裂中受hnRNP GT蛋白调控的基因和途径，并试图确定这些基因和途径是否与体细胞中受RBMX调控的基因和途径相同或不同。虽然我们不知道如果没有hnRNP GT蛋白，哪些途径会被扰乱，但我们可以从Hnrnpgt缺失小鼠的详细表型中做出强有力的预测。HnRNP GT零生殖细胞在双线期减数分裂期间停止，这是一个重要的阶段，配对的染色体刚刚开始分离。我们的初步数据显示，减数分裂染色体在Hnrnpgt缺失的睾丸中异常展开，这表明至少有一些hnRNP GT的靶标将控制减数分裂染色体结构。有趣的是，RBMX也与染色体结构有关，但其机制尚不清楚。目标3将进一步利用我们的新小鼠模型来确定hnRNP GT在减数分裂后是否具有重要功能，在此期间RBMX仍处于关闭状态。目标4将测试我们发现由小鼠RBMX和hnRNP GT控制的RNA加工通路在人类中是否保守，在不育男性中是否存在错误调节。综上所述，这些目标将剖析这个高度保守的RNA结合蛋白家族中的个体功能，以及个体蛋白质之间的表达切换如何控制基因表达的全球模式。特别是，我们将测试RBMX和hnRNP GT表达之间的切换如何有助于全球剪接程序的变化。结果。这项研究的预期结果将是更好地理解核RNA结合蛋白家族如何发挥控制发育途径的功能。这样的计划可能对整个机构都很重要。该项目将为RBMX蛋白对人类健康的重要性提供新的线索。RBMX家族基因与人类男性不育、脑和肌肉发育以及癌症有关，但仍知之甚少。这将是对这一RNA结合蛋白家族的首次全面分析。该项目的受益者将是对基因表达和发育感兴趣的科学家、我们培训的科学家和学生、不孕不育的男性以及与我们接触的公众成员。

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.