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，像人类和小鼠这样的复杂生物如何能够以相对较低的基因数量生存。嵌入前mRNA中的是控制核RNA加工的RNA结合蛋白的靶位点，因此单个基因可以编码不同的mRNA和蛋白质同种型。识别这些重要的核RNA结合蛋白的结合位点及其功能读数对于理解基因组如何工作非常重要。我们希望使用全系统的方法来帮助回答这个问题，以确定由核RNA结合蛋白Tra 2B调控的功能靶标，并发现如何通过Tra 2B与hnRNP G家族RNA结合蛋白的相互作用建立生理剪接模式。我们的研究结果将对理解剪接因子如何相互作用以灵活使用基因组中的信息以及复杂组织和专门细胞类型的发育具有重要意义。这个项目的三个方面是及时的。(1)我们最近发现，重要的人类和小鼠转录因子由Tra 2B和hnRNP G蛋白的选择性剪接调控控制（BBSRC资助的工作发表在2009年11月的PLoS Genetics和2010年1月的细胞科学杂志上）。(2)编码Tra 2B的基因的条件版本可用于该项目，它可以在任何细胞类型中失活-我们将靶向生殖细胞。(3)作为在全系统水平上全面鉴定Tra 2B调控RNA的第一步，我们已经鉴定并绘制了小鼠生殖细胞转录组内的53，000个Tra 2B结合位点。这些地点提供了丰富的受管制替代事件库，供本项目分析。我们预测这些Tra 2B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。宗旨和目标：我们将首先测试Tra 2B蛋白是否是小鼠雄性生殖细胞发育所必需的，使用条件小鼠品系，我们可以通过生殖细胞特异性共表达称为Cre重组酶的DNA切割酶来去除编码Tra 2B的基因。Tra 2是果蝇精子发生所必需的。我们将分析小鼠的睾丸结构或精子形态的缺陷，作为去除Tra 2B的结果，并纯化RNA用于分子分析。我们将使用这种RNA和我们的全转录组数据来分析Tra 2B蛋白如何调节小鼠生殖细胞中的替代mRNA亚型。我们的RNA结合数据表明，Tra 2B蛋白结合到一个'毒药外显子'含有翻译终止密码子在前mRNA的Tra 2B相关蛋白称为Tra 2A。我们将测试条件性敲除小鼠中Tra 2A毒物外显子的剪接是否改变。RNA分析将使用RT-PCR进行，我们可以快速扩增和鉴定野生型和敲除小鼠的不同mRNA亚型。由于Tra 2A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小基因的缩短基因来分析Tra 2B靶RNA，这些基因编码我们可以在细胞系中异位表达的前mRNA沿着不同水平的Tra 2B和Tra 2A。由于剪接靶以剂量依赖性方式响应于剪接调节剂的浓度，我们的预测是共表达的Tra 2B将结合到功能相关的位点，并引起剪接影响，我们将能够监测。我们将使用这些相同的小基因来测试Tra 2B与hnRNP G家族蛋白（特别是RBMY和hnRNP G-T：这两种蛋白质与Tra 2B相互作用，也涉及控制正常的人类生育力）的蛋白质相互作用的剪接效应。在我们目前的Tra 2B RNA靶点中，有一些可能影响mRNA代谢的其他方面，我们将测试Tra 2B是否具有更普遍的功能。该项目将建立规则，并提供Tra 2B蛋白调节雄性生殖系RNA加工的系统范围。

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.