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

生物学家感兴趣的一个关键问题是，像人类和老鼠这样的复杂生物体是如何在基因数量相对较少的情况下生存的。嵌入在pre- mrna中的是RNA结合蛋白的靶位点，它控制着核RNA的加工过程，因此一个基因可以编码不同的mrna和蛋白质同工型。确定这些重要的核RNA结合蛋白的结合位点，以及它们的功能读数对于了解基因组的工作原理非常重要。我们希望通过系统范围的方法来确定核RNA结合蛋白Tra2B调控的功能靶点，并通过Tra2B与hnRNP G家族RNA结合蛋白的相互作用来发现生理剪接模式是如何建立的，从而帮助回答这个问题。我们的研究结果对于理解剪接因子如何相互作用以灵活地利用基因组中的信息以及复杂组织和特殊细胞类型的发育具有重要意义。这个项目有三个方面是及时的。(1)我们最近发现重要的人和小鼠转录因子受Tra2B和hnRNP G蛋白的选择性剪接调控（BBSRC资助的研究发表在2009年11月的PLoS Genetics和2010年1月的Cell Science杂志上）。(2)编码Tra2B基因的条件版本可用于该项目，该基因可在任何细胞类型中灭活-我们将以生殖细胞为目标。(3)作为在全系统水平上全面鉴定Tra2B调控rna的第一步，我们在小鼠生殖细胞转录组中鉴定并绘制了53,000个Tra2B结合位点。这些站点为本项目提供了丰富的可选事件分析池。我们预测只有这些Tra2B结合位点的一个子集会影响剪接，我们资助的一个关键目标是确定这些位点及其功能结果。目的和目的：我们将首先测试Tra2B蛋白是否对小鼠雄性生殖细胞发育至关重要，使用条件小鼠菌株，我们可以通过生殖细胞特异性的DNA切割酶Cre重组酶的共表达去除编码Tra2B的基因。Tra2对果蝇的精子形成至关重要。我们将分析小鼠睾丸结构或精子形态的缺陷，作为去除Tra2B的结果，并纯化RNA进行分子分析。我们将使用该RNA和转录组数据来分析Tra2B蛋白如何调节小鼠生殖细胞中其他mRNA亚型。我们的RNA结合数据表明，Tra2B蛋白与Tra2A相关蛋白的前mrna中含有翻译STOP密码子的“有毒外显子”结合。我们将测试在条件敲除小鼠中，Tra2A毒性外显子的剪接是否发生变化。RNA分析将使用RT-PCR进行，我们可以快速扩增和表征野生型和基因敲除小鼠的不同mRNA亚型。由于Tra2A可能在敲除小鼠中受到影响，并提供部分冗余功能，我们还将通过使用称为minigenes的缩短基因来分析Tra2B靶rna，该基因编码的pre- mrna可以在不同水平的Tra2B和Tra2A细胞系中异位表达。由于剪接靶点对剪接调节剂的浓度有剂量依赖性的反应，我们的预测是，共表达的Tra2B将结合到功能相关的位点上，并引起剪接影响，我们将能够监测这种影响。我们将使用这些相同的小基因来测试Tra2B与hnRNP G家族蛋白相互作用的剪接效应（特别是RBMY和hnRNP G- t：这两种蛋白与Tra2B相互作用，也涉及控制正常的人类生育能力）。在我们目前的Tra2B RNA靶点中，有一些可能影响mRNA代谢的其他方面，我们将对其进行测试，以确定Tra2B是否具有更一般的功能。该项目将建立这些规则，并提供Tra2B蛋白调节雄性生殖系RNA加工的全系统图像。

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.