Elucidating mechansims and roles of alternative polyadenylation

我们的基因是由DNA组成的，但当它们被启动时，被称为RNA的相关分子中会产生拷贝，而这个RNA继续编码我们基因的蛋白质产物。当基因被复制到RNA中时，RNA被切割，并在末端添加一串腺嘌呤分子(简称A)。这种所谓的“聚A尾”起到了保护RNA不被降解的作用，有助于将RNA运送到细胞周围，并刺激RNA形成蛋白质。添加PolyA尾巴的位置并不总是相同的，即使对于相同的基因也是如此。例如，一半的人类基因含有一个以上位置的RNA，用于添加Poly A尾巴。控制PolyA尾巴的添加位置是非常重要的，因为它最终会影响基因的功能。然而，令人惊讶的是，我们对这一过程知之甚少。不只是人类的RNA有不同的聚A尾巴，其他动物和植物也是如此。我们一直在研究植物如何控制它们开花的时间，这是一个非常精确地控制基因的过程。在这项工作的过程中，我们发现了三个因素，称为FCA，FY，最近的FPA，功能控制一些RNA的PolyA位点选择。这些基因表达的基本方面在植物和动物中非常相似，事实证明，有一些人类蛋白质与FY和FPA高度相关。因此，这些蛋白质也有可能控制人类的PolyA位点选择，但人们对它们知之甚少。由于我们发现FCA和FPA并不需要对方来控制PolyA的选址，我们认为他们一定是以不同的方式来做这件事。这让我们有机会了解如何控制保利A的选址。在这项提案中，我们计划建立在我们对植物中FCA和FPA的了解的基础上，但这种知识应该更具普遍意义。我们想知道两件事：(1)FCA和FPA如何控制添加PolyA尾巴的位点(2)FCA和FPA通过控制替代PolyA位点的选择来调节哪些基因？我们将通过确定所需RNA的功能来研究FCA和FPA如何控制PolyA位点。这应该是相当简单的。我们将制作包含目标基因不同部分的测试基因，并观察它们在放回植物中时如何影响聚A位点的选择。为了找到正常的PolyA尾巴依赖于FCA和FPA的其他基因，我们将观察正常植物和缺乏FCA或FPA的突变植物中RNAs的多腺化位置。多亏了下一代测序技术，我们现在有可能查看一个细胞中几乎所有的RNA，这项技术正在为现代生物学带来革命性的变化，它为我们提供了大量的序列数据，速度非常快，成本只有以前的一小部分。这项技术已经被开发出来，通过对每一个RNA的一小部分进行测序来查看RNA，这一部分足以识别它，称为“标签”。为了找到标签，科学家们使用Poly A尾巴并对它旁边的东西进行了排序。这对我们来说是一个令人高兴的巧合，因为这意味着除了标记特定的RNA外，这种方法还告诉我们RNA中添加了聚A尾巴的位置。为了分析大量数据并进行比较，我们需要开发专门的计算工具。因为我们已经知道FCA和FPA控制PolyA位点选择的基因，如果我们的工具运行良好，我们应该能够发现这些‘标签’的变化。一旦我们确定它们是真的，我们就可以寻找“标签”中的其他变化，以确定由FCA和FPA控制的其他基因。由于许多其他科学家也在使用这种测序技术，但出于完全不同的原因，我们可以使用我们的分析工具来查看他们数据中多聚腺苷酸化的变化。通过这种方式，我们将能够确定细胞类型和情况，在那里交替多聚腺苷是基因调控的重要部分。

Our genes are made of DNA, but when they are switched on, copies are made in a related molecule called RNA and this RNA goes on to code for the protein products of our genes. As the gene is copied into RNA, the RNA is cut and a string of Adenine molecules (A for short) are added at the end. This so-called 'poly A tail' functions to protect the RNA from being degraded, and helps to transport the RNA around the cell and stimulates the formation of protein from the RNA. The site at which the poly A tail is added is not always the same, even for the same gene. For example, half of all human genes have RNAs with more than one site for adding a poly A tail. Controlling the site at which the poly A tail is added is very important because it ultimately affects how genes function. However, this is a process we know surprisingly little about. It's not just human RNAs that have different poly A tails, other animals and plants do too. We have been studying how plants control the time at which they flower, a process where genes are very precisely controlled. In the course of this work, we have discovered that three factors called FCA, FY and, most recently, FPA, function to control poly A site selection of some RNAs. Such basic aspects of gene expression are very similar in plants and animals and it turns out that there are human proteins highly related to FY and FPA. It is possible therefore, that these proteins control poly A site selection in humans too, but very little is known about them. As we have found that FCA and FPA don't need each other to control poly A site choice, we think they must be doing this in different ways. This gives us a chance to understand how poly A site choice can be controlled. In this proposal we plan to build on what we know about FCA and FPA in plants, but this knowledge should be of much more general interest. We want to know two things: (1) How do FCA and FPA control the site at which a poly A tail is added (2) What genes do FCA and FPA regulate by controlling alternative poly A site choice? We will work out how FCA and FPA control poly A sites by identifying the features of the RNA required. This should be quite straightforward. We will make test genes containing different parts of the target gene and see how they affect poly A site selection when placed back in plants. In order to find the other genes whose normal poly A tail depends on FCA and FPA, we will look at where RNAs are polyadenylated in normal plants and in mutant plants that lack FCA or FPA. It is now possible for us to look at nearly all the RNAs in a cell thanks to Next Generation Sequencing, a technology that is revolutionizing modern biology by giving us huge amounts of sequence data, very quickly and at a fraction of the cost to before. This technology has been developed to look at RNA by sequencing a short part of every RNA, sufficient to identify it, called a 'tag'. To find the tag, scientists use the poly A tail and sequence what is next to it. This is a happy coincidence for us, because it means that in addition to tagging a particular RNA, this method also tells us where a poly A tail has been added to RNA. To analyse the large amounts of data and make comparisons, we will need to develop specialized computational tools. Because we already know genes where FCA and FPA control poly A site selection, we should be able to find changes in these 'tags' if our tools are working well. Once we are sure they are, we can look for other shifts in 'tags' to identify other genes controlled by FCA and FPA. As lots of other scientists are also using this sequencing technology, but for completely different reasons, we can use our analysis tools to look at changes in polyadenylation in their data too. In this way we will be able to identify cell-types and situations where alternative polyadenylation is an important part of gene regulation.