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如何在相对较低的基因数量下生存下来。嵌入在前mRNAs中的是RNA结合蛋白的靶点，它控制着核RNA的加工，因此单个基因可以编码不同的mRNAs和蛋白质亚型。识别这些重要的核RNA结合蛋白的结合部位，并读出它们的功能，对于理解基因组的工作方式非常重要。我们想用系统级的方法来帮助回答这个问题，以确定由核RNA结合蛋白Tra2B调控的功能靶标，并发现Tra2B是如何通过与hnRNP G家族RNA结合蛋白的相互作用建立生理剪接模式的。我们的结果将对理解剪接因子如何相互作用，使基因组中的信息能够灵活使用，以及复杂组织和特殊细胞类型的发展具有重要意义。这个项目有三个方面是及时的。(1)我们最近发现，重要的人和鼠转录因子受Tra2B和hnRNP G蛋白选择性剪接调控(BBSRC资助的工作发表在2009年11月号的《PLoS遗传学》和2010年1月的《细胞科学杂志》上)。(2)编码Tra2B的基因的条件版本可用于该项目，它可以在任何类型的细胞中灭活-我们将针对生殖细胞。(3)作为在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定位了53,000个Tra2B结合位点。这些网站提供了丰富的受监管替代事件池，以供本项目分析。我们预测只有这些Tra2B结合位点的一部分会影响剪接，我们资助的一个关键目标是确定这些结合位点及其功能结果。目的和目的：我们将首先使用一个有条件的小鼠品系来测试Tra2B蛋白是否对小鼠雄性生殖细胞的发育是必要的，我们可以通过生殖细胞特异性共表达一种名为Cre重组酶的DNA切割酶来去除编码Tra2B的基因。Tra2是果蝇精子发生所必需的。我们将分析由于移除Tra2B而导致的睾丸结构或精子形态缺陷的小鼠，并提纯RNA进行分子分析。我们将使用这个RNA和我们的转录组数据来分析Tra2B蛋白如何调节小鼠生殖细胞中不同的mRNA异构体。我们的RNA结合数据表明，Tra2B蛋白与一个‘毒药外显子’结合，该外显子包含一个翻译停止密码子，位于Tra2B相关蛋白Tra2A的前mRNA中。我们将在条件性基因敲除小鼠中测试Tra2A毒物外显子的剪接是否发生变化。RNA分析将使用RT-PCR进行，利用它我们可以快速扩增和鉴定野生型和基因敲除小鼠的不同mRNA亚型。由于Tra2A可能在基因敲除小鼠中受到影响并提供部分冗余功能，我们还将使用被称为Miniges的缩短基因来分析Tra2B靶RNA，这些基因编码我们可以在不同水平的Tra2B和Tra2A的细胞系中异位表达的前-mRNAs。由于剪接靶标对剪接调节剂浓度的反应具有剂量依赖性，我们预测共表达的Tra2B将与功能相关的位点结合，并引起我们能够监测的剪接影响。我们将使用这些相同的微基因来测试Tra2B与hnRNP G家族蛋白(特别是RBMY和hnRNP G-T：这两种蛋白都与Tra2B相互作用，也参与控制正常人类生育能力)蛋白质相互作用的剪接效果。在我们目前的一组Tra2B RNA靶点中，有一些可能会影响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。该项目将制定规则，并提供一幅系统范围的图景，通过该图，Tra2B蛋白调节雄性生殖系中的RNA加工。

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.