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

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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.