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

点击翻译按钮获取中文摘要

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.