Structural and functional analysis of the splicing co-regulators PTB and Raver1

We propose to investigate how two proteins, the polypyrimidine tract binding protein (PTB) and raver1, work together to regulate the ways that genetic information is used in cells. Genes encoded in DNA are split into pieces called exons which need to be combined before the messenger RNA (mRNA) of the gene is used to make a protein. There are different ways to combine the exons, which allows several different but related proteins to be made from one gene, a process known as alternative splicing. PTB, a protein that binds RNA, helps to determine how the exons are combined in the final mRNA for a growing number of genes. For one particular gene, a-tropomyosin, PTB needs to interact with another protein, raver1, in order to control splicing. Our research aims investigate the structures PTB, raver1 and to reveal how these molecules bind to each other and to RNA. This information will help us to see at the molecular level how these proteins affect RNA structure and control how the RNA is spliced.

The polypyrimidine tract binding protein (PTB) is an RNA-binding protein containing four RRM domains that has been established as an important regulator of alternative splicing of a number of genes. In many cases PTB represses exon inclusion in the mature RNA by interacting with protein co-factors. Recent work by the Smith lab, (Cambridge University, UK) has established the regulated exon in a-tropomyosin as a particularly valuable model system for the investigation of the molecular mechanism PTB since they have shown that it needs to interact with the co-factor raver1 for effective splicing repression. In our previous work we have determined the solution structures of all four RRM domains of PTB. More recently, as part of an ongoing collaboration with the Smith group, we have provided the first structural insights into PTB-raver1 interactions by showing how a PTB-binding peptide from raver1 interacts with the dorsal surface of PTB-RRM2. Despite these important advances, we are still a long way from understanding how PTB and raver1 co-operate to regulate splicing. We need to develop more sophisticated structural analyses of functional complexes in order to shed new light on PTB-regulated splicing. In this new proposal we aim to determine the structure of a high-affinity PTB-raver1 complex and the structures of protein-RNA complexes for both proteins. This is a timely study that is built on a great deal of previous work and is now poised to yield important new insights into the role of PTB in alternative splicing. We will use a combination of X-ray crystallographic, NMR and small-angle X-ray scattering approaches for these structure determinations, applying the most appropriate technique for each complex. In each case the structural studies will be preceded by careful investigation of the protein and RNA components required for high-affinity interactions. In addition to laying the necessary groundwork, these binding studies will in themselves provide new insights