权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Regulation of alternative splicing by G-quadruplexes: molecular mechanisms and tools to manipulate gene expression

G-四链体对选择性剪接的调控：操纵基因表达的分子机制和工具

基本信息

批准号：
BB/R006555/1
负责人：
Ian EPERON
金额：
$ 92.64万
依托单位：
University of Leicester
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2018
资助国家：
英国
起止时间：
2018 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FR006555%2F1
关键词：
Regulation alternative splicing quadruplexes molecular

项目摘要

When RNA polymerase starts to transcribe a gene into mRNA, the sequence and thus the activity of the protein encoded by the RNA depend on the pattern in which large portions of the RNA are spliced out. The processes by which the sites of splicing are selected are very complex, and they are still understood poorly. Understanding them is hugely important both because splicing is an essential process that, more than anything else, enables highly complex organisms such as ourselves to have developed despite having only the same number of genes as much simpler organisms and because, by controlling splicing, we could shift the expression of a gene from one type of protein to another for therapeutic purposes. Indeed, the first drugs targeting splicing in muscle and the CNS have been approved recently by the US FDA and others are in trials. There has been great excitement recently over the discovery that quadruplexes (G4s) might regulate splicing. G4s are small, four-stranded structures that can form in the RNA from four sequences of GG or GGG in close proximity. They could open up new ways of understanding and manipulating splicing. However, it has been very difficult to prove that they form in long RNA molecules in functional splicing conditions, and nothing is known of how they might affect splicing. We have recently published a new method, called FOLDeR, that enables us to map the regions of a pre-mRNA that form G4s in splicing conditions. We have applied this to Bcl-X, a gene expressing two isoforms of protein: one promotes cell survival and the other promotes apoptosis. The difference results from the choice between two 5' splice sites. We have shown that there are two G4-forming sequences in Bcl-X, one close to each splice site.Many small molecules are known to bind to and stabilize G4s. We have tested a range of 33 of these on Bcl-X. Both in nuclear extracts and in cells, one reagent shifts splicing so much that the usually minor pro-apoptotic isoform becomes predominant, and we have shown that it affects the structures of the two G4-forming regions in the RNA, probably by enhancing G4 formation. Moreover, it switches the splicing of another gene crucial for some cancers, Mcl-1, to express only the pro-apoptotic isoform. Accordingly, it promotes apoptosis. The same reagent has no effects on some other alternative splicing events, and others that affect different alternative splicing events have no effects on Bcl-X. Most of the other 32 compounds show mild or no effects. Importantly, the effects of each one on Bcl-X splicing are similar in nuclear extracts with purified pre-mRNA and in cells, showing that the molecules affect splicing directly. This suggests that G4 stabilizers might each target a defined set of genes. Are these genes in sets with common biological functions? If not, could we investigate how the small molecules and their cognate G4s work so that we can prevent unwanted effects and develop useful and selective drugs? Could we predict the sites of action of such molecules? Could we use their target sequences to develop ligand dependent splicing switches, enabling a gene to switch from one function to another?We propose to address these exciting possibilities using four approaches. (i) The first is use high-throughput sequencing to identify all the changes in expression and splicing brought about by several G4-binding molecules in cells, which will inform us about the range of effects and the common features associated with the targets of each molecule. (ii) We will identify and test the exact nucleotides and contacts associated with G4 formation and small molecule binding by methods including NMR and X-ray crystallography. (iii) We will synthesize and test a range of new analogues to help in defining interactions and, using structural information, to improve selectivity. (iv) We will determine how G4s affect splicing and use this to test whether G4s can be inserted as switches into new positions.

当RNA聚合酶开始将基因转录成mRNA时，由RNA编码的蛋白质的序列和活性取决于大部分RNA被剪接的模式。选择剪接位点的过程非常复杂，人们对它们的了解仍然很少。了解它们非常重要，因为剪接是一个重要的过程，比其他任何事情都重要，使我们这样的高度复杂的生物体能够发展，尽管只有与简单得多的生物体相同数量的基因，因为通过控制剪接，我们可以将基因的表达从一种蛋白质转移到另一种蛋白质，以达到治疗目的。事实上，第一批靶向肌肉和中枢神经系统剪接的药物最近已被美国FDA批准，其他药物正在试验中。最近，四链体（G4）可能调节剪接的发现引起了极大的兴奋。G4是一种小的四链结构，可以在RNA中由四个非常接近的GG或GGG序列形成。它们可以开辟理解和操纵剪接的新途径。然而，很难证明它们在功能性剪接条件下形成于长RNA分子中，也不知道它们如何影响剪接。我们最近发表了一种名为FOLDeR的新方法，使我们能够绘制在剪接条件下形成G4的前mRNA区域。我们将其应用于Bcl-X，该基因表达两种蛋白质亚型：一种促进细胞存活，另一种促进细胞凋亡。这种差异是由两个5'剪接位点之间的选择引起的。我们已经发现Bcl-X中有两个G4形成序列，一个靠近一个剪接位点。我们已经在Bcl-X上测试了其中的33种。无论是在核提取物和细胞中，一种试剂的剪接移位如此之多，通常是次要的促凋亡亚型成为主要的，我们已经表明，它会影响RNA中的两个G4形成区域的结构，可能是通过增强G4的形成。此外，它还改变了另一个对某些癌症至关重要的基因Mcl-1的剪接，使其只表达促凋亡亚型。因此，它促进细胞凋亡。相同的试剂对其他一些选择性剪接事件没有影响，而其他影响不同选择性剪接事件的试剂对Bcl-X没有影响。其他32种化合物中的大多数显示出轻微或无影响。重要的是，每一个对Bcl-X剪接的影响在具有纯化的前mRNA的核提取物和细胞中是相似的，表明这些分子直接影响剪接。这表明G4稳定剂可能各自靶向一组确定的基因。这些基因是否具有共同的生物学功能？如果没有，我们是否可以研究小分子及其同源G4如何工作，以便我们可以防止不必要的影响，并开发有用的和选择性的药物？我们能预测这些分子的作用位点吗？我们能否利用它们的靶序列来开发配体依赖性剪接开关，使基因从一种功能切换到另一种功能？我们建议使用四种方法来解决这些令人兴奋的可能性。(i)第一个是使用高通量测序来鉴定细胞中几种G4结合分子所带来的表达和剪接的所有变化，这将告知我们与每个分子的靶点相关的作用范围和共同特征。(ii)我们将通过NMR和X射线晶体学等方法鉴定和测试与G4形成和小分子结合相关的确切核苷酸和接触。(iii)我们将合成和测试一系列新的类似物，以帮助定义相互作用，并使用结构信息，以提高选择性。(iv)我们将确定G4如何影响剪接，并使用它来测试G4是否可以作为开关插入到新的位置。