Deciphering the functions of the RNA binding protein T-STAR in mouse development

RNA结合蛋白在组织细胞中的基因表达中起关键作用，并控制选择性剪接的重要过程，通过该过程，单个基因可以产生多种不同的mRNA和蛋白质。大多数基因产生选择性剪接的mRNA，并且一些个体基因也产生许多剪接异构体。在哺乳动物中最常见的选择性剪接中，整个外显子要么被剪接到mRNA中，要么被排除在mRNA之外。替代外显子的插入对于下游蛋白质功能可能非常重要。选择性剪接受核RNA结合蛋白控制。许多核RNA结合蛋白以密切相关蛋白质的家族形式存在，但家族成员是否具有不同或重叠的功能通常是一个谜。一个很好的例子是T-STAR RNA结合蛋白，它与Sam 68属于同一家族。T-STAR和Sam 68对正常健康很重要。T-STAR基因在成神经管细胞瘤的某些病例中被扩增，而Sam 68在严重的神经退行性疾病脆性X震颤相关共济失调中被隔离。T-STAR和Sam 68在转染细胞中的功能相似，以调节相同靶外显子的剪接。我们希望确定内源性T-STAR蛋白的作用，以及它是否与Sam 68相互作用以控制体内剪接。本项目的三个方面是及时的：（1）我们最近做了一个T-STAR空鼠标用于本项目。这里有一个机会之窗，我们是全球唯一一个拥有T-STAR null小鼠的组织（我们已经将我们的小鼠提交给欧洲小鼠突变体档案馆，明年8月发布）。(2)在使用强大的高通量方法的初步数据中，我们已经确定了基因敲除小鼠中的剪接缺陷。我们发现，在小鼠大脑中，由T-STAR控制的四个已经确定的替代外显子包括Neurexin基因中的替代外显子，该基因编码神经系统布线所必需的蛋白质。Neurexin基因在野生型大脑中具有区域剪接模式，但这种区域控制在T-STAR蛋白不存在的情况下完全破坏。这表明T-STAR是控制这种区域剪接模式的主调节因子。(3)通过与罗马的Sette小组的国际合作，我们获得了一个Sam 68无效等位基因以与我们的T-STAR无效小鼠杂交。因此，我们将能够分析表型的变化，并确定双敲除小鼠（缺失Sam 68和T-STAR）中的剪接缺陷。在目前的项目中，我们将全面鉴定T-STAR靶RNA，以发现T-STAR是否主要调节突触蛋白或负责建立大脑中的区域剪接模式（或两者兼而有之）。我们将确定T-STAR和Sam 68在控制某些靶外显子时是否具有冗余功能，以及它们在大脑中的表达重叠程度。我们将绘制T-STAR结合RNA的位置，并找出这种结合调节选择性剪接的重要特征。我们将研究当通过去除T-STAR阻断选择性剪接事件时小鼠的表型后果。最后，我们将讨论T-STAR和Sam 68是否冗余地控制重要的发育步骤，特别是在睾丸中，这两种蛋白质都以高水平表达，以及大脑。我们预计，该项目的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大脑和专门细胞类型等复杂组织的发育具有重要意义。我们的项目将发现剪接控制的新的调节靶点，询问调节机制和当其被阻断时的表型后果。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。

RNA binding proteins play a key role in organising gene expression in the cell, and control the important process of alternative splicing through which a single gene can produce multiple different mRNAs and proteins. Most genes produce alternatively spliced mRNAs, and some individual genes also produce many splice isoforms. In the most common kind of alternative splicing in mammals entire exons are either spliced in or left out of the mRNA. The insertion of alternative exons can be very important for downstream protein function. Alternative splicing is controlled by nuclear RNA binding proteins. Many nuclear RNA binding proteins exist as families of closely related proteins, but whether family members have different or overlapping functions is usually a mystery. A good example of this is the T-STAR RNA binding protein, which is in the same family as Sam68. T-STAR and Sam68 are important for normal health. The T-STAR gene becomes amplified in some cases of medulloblastoma, and Sam68 becomes sequestered in the severe neurodegenerative disorder Fragile X Tremor Associated Ataxia. T-STAR and Sam68 function similarly in transfected cells to regulate splicing of the same target exons. We want to establish the roles of endogenous T-STAR protein and if it interacts with Sam68 to control splicing in vivo. Three aspects of this project are timely: (1) We have recently made a T-STAR null mouse to use in the current project. There is a window of opportunity here where we are the only group worldwide with a T-STAR null mouse (we have submitted our mouse to the European Mouse Mutant Archive for release next August). (2) In preliminary data using a powerful high throughput approach we have identified splicing defects in the knockout mice. The four already identified alternative exons which we have found to be controlled by T-STAR in the mouse brain include alternative exons in the Neurexin genes which encode proteins essential for wiring up the nervous system. The Neurexin genes have regional splicing patterns in the wild type brain, but this regional control totally breaks down in the absence of T-STAR protein. This indicates that T-STAR is the master regulator controlling this regional splicing pattern. (3) Through international collaboration with the Sette group in Rome we have access a Sam68 null allele to cross in to our T-STAR null mouse. Hence we will be able to analyse changes in phenotype and identify splicing defects in double knockout mice (missing Sam68 and T-STAR).Aims and objectives. In this current project we will comprehensively identify T-STAR target RNAs to discover if T-STAR predominantly regulates synaptic proteins or is responsible for establishing regional patterns of splicing in the brain (or both). We will establish if T-STAR and Sam68 function redundantly in the control of some target exons, and to what extent they overlap in expression in the brain. We will map where T-STAR binds RNA and work out the important features through which this binding regulates alternative splicing. We will examine the phenotypic consequences for the mouse when alternative splicing events are blocked by removing T-STAR. Finally we will address whether T-STAR and Sam68 work redundantly to control important developmental steps, particularly in the testis where both proteins are expressed at high levels as well as the brain. We expect that the results of this project will be significant in understanding how splicing factors interact to enable flexible use of information in the genome and the development of complex tissues like the brain and specialised cell types. Our project will discover new regulated targets of splicing control, 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.