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

研究目的：RNA结合蛋白如Tra2b在组织基因表达中起关键作用。这包括选择性剪接过程，通过该过程，大多数单个基因产生多个mRNA剪接异构体。选择性剪接在构建复杂的动物身体中起着关键作用。由于Tra2b是小鼠中整个动物生存所必需的，因此与其在癌细胞中的作用相比，它在健康动物中调节的全局靶点和途径要少得多。这项研究的目的是解决这一知识空白的遗传温顺的生殖细胞发育途径，使精子，并涉及细胞分裂有丝分裂和减数分裂和细胞分化。时间性和金钱的价值：这个项目的四个方面是特别及时。1)我们有一个现有的条件性Tra2b敲除小鼠，我们可以在生殖细胞（在发育途径导致精子）。生殖细胞发育在成年小鼠中非常活跃，但对生存能力并不重要。我们的初步工作表明，Tra2b对于一组重要的生殖细胞（称为生殖细胞，它们是所有其他下游生殖细胞发育阶段的前体）的生存至关重要。2)通过与美国的世界领先小组的国际合作，我们将能够在转基因生殖细胞中识别Tra2b剪接靶点。这项合作已经使我们能够确定生殖细胞内Tra2b的初始剪接靶点，并将在项目后期帮助我们。3)我们已经使用交联和下一代测序确定了数百万个Tra2b的RNA结合位点。4)Harwell的MRC单位正在制作一个小鼠模型，使我们能够探测剪接反馈途径的功能，该途径控制Tra2b的表达水平，并在睾丸中上调。这个模型将在以后广泛使用，但有一个机会窗口，我们将有唯一的访问，并可以做出真正重要的贡献，以了解为什么这个反馈途径是重要的。目标和目的：这个项目将破译Tra2b的剪接活动如何控制生殖细胞发育。我们的第一个目标将是确定由Tra2b在生殖细胞中控制的剪接靶点，并分析这些靶点以确定对生殖细胞存活重要的基因表达途径。我们的第二个目标将是在减数分裂期间确定Tra2b的靶点。我们希望这样做，因为Tra2b在减数分裂中上调。我们的RNA结合数据预测Tra2b控制重要的减数分裂基因，并预测我们想要在全球范围内测试的Tra2b剪接控制机制。我们的第三个目标是测试Tra2b剪接活动在减数分裂过程中是如何控制的剪接反馈控制途径。这种剪接反馈控制涉及基因组的超保守区域，表明它非常重要，并且在减数分裂细胞中非常活跃，但尚未在动物模型中对其进行功能研究。我们预测，这种反馈控制途径可能会稳定Tra2b控制减数分裂剪接的正常模式，或者可能会阻止Tra2b水平增加到可能损害转录组的程度。结果：我们希望该项目的结果将对理解剪接因子如何发挥作用，以灵活利用基因组中的信息，睾丸等复杂组织的发育，和特殊的细胞类型，比如那些正在经历减数分裂的细胞。我们的项目将发现剪接控制的新的调节靶点，并询问当其被阻断时的调节机制和表型后果。这项工作的主要受益者将是对基因表达感兴趣的科学家、将接受培训的科学家和学生以及我们将参与的公众。

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.