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

当RNA聚合酶开始将一个基因转录成mRNA时，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。了解它们是非常重要的，因为剪接是一个重要的过程，比其他任何事情都重要，它使像我们这样高度复杂的生物体得以发展，尽管只有与更简单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，第一批针对肌肉和中枢神经系统剪接的药物最近已获得美国食品和药物管理局的批准，其他药物正在试验中。最近有一项非常令人兴奋的发现，四联体（G4s）可能调节剪接。G4s是一种小的四链结构，可以在RNA中由GG或GGG的四个序列紧密相连形成。它们可以开辟理解和操纵拼接的新途径。然而，很难证明它们在功能性剪接条件下在长RNA分子中形成，并且对它们如何影响剪接一无所知。我们最近发表了一种名为FOLDeR的新方法，它使我们能够绘制出在剪接条件下形成G4s的前mrna区域。我们将此应用于Bcl-X，一种表达两种同种异构体蛋白质的基因：一种促进细胞存活，另一种促进细胞凋亡。这种差异源于两个5'剪接位点之间的选择。我们已经证明在Bcl-X中有两个g4形成序列，一个靠近每个剪接位点。已知许多小分子可以结合并稳定G4s。我们已经在Bcl-X上测试了33个。在核提取物和细胞中，一种试剂将剪接改变得如此之多，以至于通常较小的促凋亡同种异构体变得占优势，我们已经证明它影响RNA中两个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，使其只表达促凋亡同种异构体。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表现出轻微或没有影响。重要的是，它们对Bcl-X剪接的影响在纯化前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稳定剂可能分别针对一组特定的基因。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G4s如何起作用，以便我们可以防止不必要的影响并开发有用和选择性的药物？我们能预测这些分子的作用位置吗？我们能否利用它们的靶序列来开发依赖于配体的剪接开关，从而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)第一种是利用高通量测序技术鉴定细胞中几种g4结合分子所带来的表达和剪接的所有变化，从而了解每种分子的作用范围和与靶点相关的共同特征。（ii）我们将通过核磁共振和x射线晶体学等方法识别和测试与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，以帮助确定相互作用，并利用结构信息提高选择性。（iv）我们将确定G4s如何影响拼接，并以此来测试G4s是否可以作为开关插入到新的位置。

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.