Mapping PTB interactions with its RNA targets to elucidate its role in activating cellular IRESs and regulating pre-mRNA splicing

The characteristics of every cell in an organism (and hence the characteristics of the whole organism) are determined by the spectrum of proteins present. Each of these proteins is specified in the genome, the DNA of the organism. The process of generating a particular type of protein involves copying the relevant section of DNA into messenger RNA (mRNA), which is then decoded to give rise to the protein. Thus an mRNA can be regarded as equivalent to a video tape, which can be decoded by an appropriate device (video recorder) to give pictorial information, and the DNA can be regarded as an archive of all possible videos, joined together end-to-end. However, in mammals and other higher organisms, the relevant section of the DNA is initially copied into a pre-mRNA which needs to be processed to give the mature mRNA suitable for decoding. In the video analogy, this processing involves splicing out irrelevant parts of the pre-mRNA tape, and splicing the remaining parts together to give a video tape suitable for decoding. Remarkably, most of the pre-mRNA video tapes in mammalian organisms can be spliced in more than one way, resulting in a number of slightly different versions of the final video. This project concerns a protein known as polypyrimidine tract binding protein (PTB) which plays a major role in determining which pattern of alternative splicing will occur. In addition, it also plays an important role in the decoding of certain mRNAs. PTB is a protein which binds to (interacts with) RNA, showing preference for specific RNA sequences. PTB actually has four distinct RNA binding surfaces, so that a single PTB molecule can interact simultaneously with 4 different segments of the RNA. It is thought that this multi-site binding is the key to how PTB can regulate the pattern of splicing, and promote decoding of certain mRNAs. We have developed methods to determine the orientation of PTB binding to any RNA (i.e. which of the four RNA binding surfaces binds to which site on the RNA), and to find out which of these four PTB-RNA interactions is critical for the biological action of PTB. We will use these methods to examine (i) how PTB regulates the decoding of three different mRNAs that are known to require PTB binding in order that decoding can be initiated, and (ii) how PTB regulates the pattern of alternative splicing of three different pre-mRNAs.

Under a current BBSRC Project Grant (BB/E004857/1), we have prepared a library of PTB mutants for use in tethered hydroxyl radical footprinting assays to map the orientation of PTB binding to its RNA targets (i.e. which RBD binds to which site on the RNA), and we have validated the approach and the particular mutants by mapping PTB interactions with two picornavirus IRESs. We are also part way through constructing PTB mutants in which the RNA binding potential of each RBD has been inactivated, to enable us to determine which RBD/target RNA interactions are critical for the biological activity of PTB. In this proposed project, these reagents will be used to examine how PTB activates certain cellular mRNA IRESs, and how it regulates splicing of certain pre-mRNAs that are subject to alternative splicing. For each type of RNA target, the orientation of PTB binding will be determined using our library of mutants in tethered hydroxyl radical footprinting assays, and the requirement for each RBD/target RNA interaction for the in vitro biological activity of the PTB will be assessed using the four mutants in which an individual RBD has been inactivated. The cellular mRNA IRESs to be studied will be c-myb, BAG-1(S) and Apaf-1. These have been chosen on the grounds that the internal ribosome 'landing site' has already been mapped, the PTB binding sites are known, and IRES activity is stimulated by PTB in an in vitro system. The pre-mRNA substrates subject to PTB-regulated alternative splicing will be the tropomyosin1 exon 3, FAS exon 6, and, if time allows, the N1 exon of c-src. These have likewise been chosen on the grounds that the regulation of splicing by PTB can be recapitulated in an in vitro system, and potential PTB binding sites are already known. The outcome of this work will be a greater understanding of how PTB activates the selected cellular IRESs, and regulates the splicing of the optional exon in the chosen pre-mRNA targets.