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

生物体中每个细胞的特性（以及整个生物体的特性）都是由存在的蛋白质谱决定的。这些蛋白质中的每一种都在基因组中被指定，也就是生物体的DNA。产生一种特定类型蛋白质的过程包括将DNA的相关部分复制到信使RNA （mRNA）中，然后对信使RNA进行解码以产生这种蛋白质。因此，mRNA可以被视为相当于一盘录像带，可以通过适当的设备（视频记录器）解码以提供图像信息，而DNA可以被视为所有可能的视频的存档，端到端连接在一起。然而，在哺乳动物和其他高等生物中，DNA的相关部分最初被复制成一个pre-mRNA，这个pre-mRNA需要经过加工才能形成适合解码的成熟mRNA。在视频的类比中，这种处理包括将pre-mRNA磁带的不相关部分拼接出来，并将剩余部分拼接在一起，从而得到适合解码的视频磁带。值得注意的是，在哺乳动物生物体中，大多数pre-mRNA视频磁带可以以多种方式拼接，从而产生许多略有不同的最终视频版本。该项目涉及一种被称为多嘧啶束结合蛋白（PTB）的蛋白质，它在决定将发生哪种选择性剪接模式方面起着重要作用。此外，它在某些mrna的解码中也起着重要作用。PTB是一种与RNA结合（相互作用）的蛋白质，对特定的RNA序列表现出偏好。PTB实际上有四个不同的RNA结合表面，因此一个PTB分子可以同时与4个不同的RNA片段相互作用。据认为，这种多位点结合是PTB如何调节剪接模式并促进某些mrna解码的关键。我们已经开发出方法来确定PTB与任何RNA结合的方向（即四种RNA结合表面中的哪一种与RNA上的哪个位点结合），并找出这四种PTB-RNA相互作用中哪一种对PTB的生物作用至关重要。我们将使用这些方法来研究(i) PTB如何调节三种不同mrna的解码，这些mrna已知需要PTB结合才能启动解码，以及（ii） PTB如何调节三种不同pre- mrna的选择性剪接模式。

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.