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

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序列已经被计算出来，人类蛋白质编码基因在2 -3万个之间。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了基因的数量。现在一个重要的问题是细胞如何弥补数量上的差距。一种重要的方法似乎是使用同一基因编码多种蛋白质。1993年的诺贝尔奖授予了一项重要发现，即人类等生物的基因被分成编码蛋白质的部分（称为外显子）和非编码区域（称为内含子）。DNA被复制成RNA，而RNA又被用来制造蛋白质。RNA合成后，外显子在细胞内结合，通过去除内含子得到编码蛋白质的模板。来自同一基因的RNA通常包含不同的外显子组合，从而导致变异。例如，有时一些外显子和内含子一起被移除。这个过程（称为选择性剪接）在发育过程中至关重要，甚至可能是允许多细胞发育的重要进化步骤。尽管如此，它还没有像控制（转录）那样被研究得多，控制（转录）首先决定哪些基因被打开和关闭来制造RNA。选择性RNA剪接是由与细胞核内RNA结合的蛋白质控制的。其中一些蛋白质不是在身体的每个部位都产生，而是只在大脑或睾丸等特定组织中产生。迄今为止的证据表明，这些因素可能发挥着非常重要的作用。其中一种被称为T-STAR的RNA结合蛋白引起了人们的特别关注，因为它可能在发育过程中与RNA加工甚至转录的信号通路的剪接和连接中发挥作用。研究人类基因功能的一个好方法是观察相应的小鼠基因，我们建议测试T-STAR在小鼠发育中的作用。和人类一样，老鼠也有一种T-STAR基因，这种基因在成年后的睾丸、大脑和肾脏发育过程中被激活。尽管小鼠和人类的T-STAR蛋白几乎相同，但它们的重要区别在于它们的调节方式不同。因此，我们预测T-STAR蛋白在人类和小鼠中调节相同基因的方式不同，这可能有助于解释小鼠与人类不同的一些原因。我们将在老鼠身上制造条件版的T-STAR基因。接下来，当我们想要的时候，我们可以通过从染色体上切下它的一个重要部分来使这个条件T-STAR失活。我们的方法是将老鼠与一种特殊的老鼠交配，这种老鼠会表达另一种叫做“重组酶”的蛋白质。我们将首先打开小鼠体内每个细胞中的重组酶，从每个细胞中去除T-STAR。我们预计会在大脑、肾脏和生殖细胞中发现缺陷，但也有可能这些小鼠在发育过程中死亡。因此，我们还将选择性地切除睾丸（制造精子的地方）中的T-STAR。这是成人T-STAR表达的主要部位，它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身上，它不是必需的。这些老鼠可能无法生育，但它们不会死。因此，通过去除组织中的T-STAR，即使我们在发育中遇到障碍，我们也可以在成年小鼠中分析这一点并获得突变细胞。我们将分析这些小鼠的基因表达，看看是否表达了不同的基因，以及转录本是否包含与不包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，我们将比较小鼠和人类中受T-STAR缺失影响的基因的转录和剪接模式。

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..