A transgenic approach to investigate the RNA binding protein T-STAR

研究 RNA 结合蛋白 T-STAR 的转基因方法

• 批准号: BB/D013917/1
• 负责人: David Elliott
• 金额: $ 47.53万
• 依托单位: Newcastle University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2006
• 资助国家: 英国
• 起止时间: 2006 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FD013917%2F1
• 关键词: transgenic; approach; investigate; RNA; binding

Human genes are found on chromosomes, and encoded by DNA. Recently the almost complete DNA sequence of humans has been worked out, and there are between 20-30 thousand human protein coding genes. Although this is a big number, most recent estimates have shown that the protein number in human cells actually far exceeds the number of genes. An important question has now become how does the cell bridge the gap in numbers. One important way seems to be to use the same gene to encode more than one protein. The 1993 Nobel Prize was awarded for the important discovery that the genes of organisms like humans are split between bits that encode proteins (called exons) separated by non-coding regions (called introns). DNA is copied into RNA which in turn is used to make protein. After RNA is made, exons are joined together in the cell, by removing introns to give the template which encodes protein. Frequently different exon combinations are included into RNA from the same gene, resulting in variation. For instance, sometimes some of the exons are removed along with the introns. This process (called alternative splicing) is critically important in development, and might even have been an important evolutionary step in allowing the development of multicellularity. Despite this, it has not been studied as much as the controls (transcription) which decide which genes are turned on and off to make the RNA in the first place. Alternative RNA splicing is controlled by proteins which bind to RNA in the nucleus. Some of these proteins are not made in every part of the body but only in particular tissues such as the brain or the testis. Evidence so far suggests that these are likely to have very important roles. One of these RNA binding proteins, called T-STAR, is of particular interest since it may play roles in splicing and connecting signalling pathways with RNA processing and possibly even transcription during development. A good way of investigating the function of a human gene is to look at the equivalent mouse gene, and we propose to test the role of T-STAR in mouse development. Mice, like humans, have a T-STAR gene which is turned on in the adult testis, developing brain and kidney. Although mouse and human T-STAR proteins are virtually identical, they have an important difference in that they are regulated differently. For this reason we predict that T-STAR protein will regulate the same genes differently in humans and mice, and this might help explain some of the reasons mice are different from humans. We will make a conditional version of the T-STAR gene in mice. Next, we can inactivate this conditional T-STAR by cutting an important part of it out of the chromosome when we want to. The way we do this is by mating the mice with special mice which express another protein called a 'recombinase'. We will first remove T-STAR from every cell in the mouse body by turning on the recombinase in every cell. We expect to see defects in the brain, kidney and germ cells,but it could be that these mice will die while they are developing . For this reason, we will also selectively remove T-STAR in the testis (where the sperm are made). This is the main site of T-STAR expression in the adult, and it is an unusual organ because you can see all the major stages of sperm development occurring in the adult and it is non-essential. The mice might be infertile but they will not die. Hence by removing T-STAR in this tissue, even if we get a block in development we will be able to analyse this in the adult mouse and obtain mutant cells. We will analyse gene expression in these mice to see if different genes are expressed, and if transcripts contain different exons from mice which do not contain the deletion. To find out if these mouse genes are regulated differently in humans, we will then compare transcription and splicing patterns of genes affected by T-STAR deletion in the mouse with their human counterparts..

人类基因存在于染色体上，由DNA编码。最近，人类几乎完整的DNA序列已被确定，人类蛋白质编码基因约有2 - 3万个。虽然这是一个很大的数字，但最近的估计表明，人类细胞中的蛋白质数量实际上远远超过基因的数量。现在一个重要的问题已经成为细胞如何弥合数量上的差距。一个重要的方法似乎是使用同一个基因编码多种蛋白质。1993年的诺贝尔奖是因为一项重要发现而获得的，即像人类这样的生物体的基因被分成编码蛋白质（称为外显子）的比特，这些比特被非编码区域（称为内含子）分开。DNA被复制成RNA，RNA又被用来制造蛋白质。RNA合成后，外显子在细胞中通过去除内含子连接在一起，得到编码蛋白质的模板。通常，不同的外显子组合包含在来自同一基因的RNA中，导致变异。例如，有时一些外显子与内含子一起被沿着移除。这个过程（称为选择性剪接）在发育中至关重要，甚至可能是允许多细胞发育的重要进化步骤。尽管如此，它还没有被研究得像控制（转录）一样多，控制（转录）决定哪些基因被打开和关闭以首先制造RNA。选择性RNA剪接由与细胞核中的RNA结合的蛋白质控制。这些蛋白质中的一些并不是在身体的每个部位都产生，而是仅在特定的组织中产生，如大脑或睾丸。迄今为止的证据表明，这些可能具有非常重要的作用。其中一种RNA结合蛋白，称为T-STAR，特别令人感兴趣，因为它可能在剪接和连接信号通路与RNA加工，甚至可能在发育过程中转录中发挥作用。研究人类基因功能的一个好方法是观察小鼠的等效基因，我们建议测试T-STAR在小鼠发育中的作用。小鼠和人类一样，有一个T-STAR基因，该基因在成年睾丸中开启，发育大脑和肾脏。虽然小鼠和人类的T-STAR蛋白几乎是相同的，但它们有一个重要的区别，即它们的调节方式不同。因此，我们预测T-STAR蛋白质将在人类和小鼠中以不同的方式调节相同的基因，这可能有助于解释小鼠与人类不同的一些原因。我们将在小鼠中制造T-STAR基因的条件版本。接下来，我们可以通过在我们想要的时候从染色体上切下它的一个重要部分来删除这个条件T-STAR。我们的方法是让这些小鼠与表达另一种称为“重组酶”的蛋白质的特殊小鼠交配。我们将首先通过打开每个细胞中的重组酶来从小鼠体内的每个细胞中去除T-STAR。我们预计会看到大脑、肾脏和生殖细胞的缺陷，但这些小鼠可能会在发育过程中死亡。出于这个原因，我们还将选择性地去除睾丸（精子产生的地方）中的T-STAR。这是T-STAR在成年人中表达的主要部位，这是一个不寻常的器官，因为你可以看到精子发育的所有主要阶段都发生在成年人中，它是不必要的。老鼠可能会不育，但它们不会死。因此，通过去除该组织中的T-STAR，即使我们在发育中受到阻碍，我们也能够在成年小鼠中分析并获得突变细胞。我们将分析这些小鼠的基因表达，看看是否有不同的基因表达，以及转录本是否含有与不含缺失的小鼠不同的外显子。为了找出这些小鼠基因在人类中是否受到不同的调控，我们将比较小鼠中受T-STAR缺失影响的基因的转录和剪接模式与人类对应基因。

项目成果

Human Tra2 proteins jointly control a CHEK1 splicing switch among alternative and constitutive target exons.

• DOI: 10.1038/ncomms5760
• 发表时间: 2014-09-11
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Best, Andrew;James, Katherine;Dalgliesh, Caroline;Hong, Elaine;Kheirolahi-Kouhestani, Mahsa;Curk, Tomaz;Xu, Yaobo;Danilenko, Marina;Hussain, Rafiq;Keavney, Bernard;Wipat, Anil;Klinck, Roscoe;Cowell, Ian G.;Lee, Ka Cheong;Austin, Caroline A.;Venables, Julian P.;Chabot, Benoit;Koref, Mauro Santibanez;Tyson-Capper, Alison;Elliott, David J.
• 通讯作者: Elliott, David J.

Expression and functions of the star proteins Sam68 and T-STAR in mammalian spermatogenesis.

• DOI: 10.1007/978-1-4419-7005-3_5
• 发表时间: 2010
• 期刊: Advances in experimental medicine and biology
• 作者: Ingrid Ehrmann;D. Elliott

The tissue-specific RNA binding protein T-STAR controls regional splicing patterns of neurexin pre-mRNAs in the brain.

• DOI: 10.1371/journal.pgen.1003474
• 发表时间: 2013-04
• 期刊: PLoS genetics
• 影响因子: 4.5
• 作者: Ehrmann I;Dalgliesh C;Liu Y;Danilenko M;Crosier M;Overman L;Arthur HM;Lindsay S;Clowry GJ;Venables JP;Fort P;Elliott DJ
• 通讯作者: Elliott DJ

Illuminating the Transcriptome through the Genome.

• DOI: 10.3390/genes5010235
• 发表时间: 2014-03-14
• 期刊: Genes
• 影响因子: 3.5
• 作者: Elliott DJ

A SLM2 Feedback Pathway Controls Cortical Network Activity and Mouse Behavior.

• DOI: 10.1016/j.celrep.2016.12.002
• 发表时间: 2016-12-20
• 期刊: Cell reports
• 影响因子: 8.8
• 作者: Ehrmann I;Gazzara MR;Pagliarini V;Dalgliesh C;Kheirollahi-Chadegani M;Xu Y;Cesari E;Danilenko M;Maclennan M;Lowdon K;Vogel T;Keskivali-Bond P;Wells S;Cater H;Fort P;Santibanez-Koref M;Middei S;Sette C;Clowry GJ;Barash Y;Cunningham MO;Elliott DJ
• 通讯作者: Elliott DJ