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)是由存在的蛋白质的光谱决定的。这些蛋白质中的每一种都在基因组中指定，也就是生物体的DNA。产生特定类型蛋白质的过程包括将DNA的相关片段复制到信使RNA(信使RNA)中，信使RNA随后被解码以产生蛋白质。因此，一条mRNA可以被视为等同于一盘录像带，它可以被适当的设备(录像机)解码以提供图像信息，而DNA可以被视为所有可能的视频的档案，端到端地连接在一起。然而，在哺乳动物和其他高等生物中，DNA的相关片段最初被复制到前信使核糖核酸中，需要进行处理才能获得适合解码的成熟信使核糖核酸。在视频类比中，这一过程包括拼接出前mRNA磁带的不相关部分，并将其余部分拼接在一起，得到适合解码的录像带。值得注意的是，哺乳动物生物体中的大多数前信使核糖核酸录像带可以以多种方式拼接，导致最终视频的一些略有不同的版本。这个项目涉及一种被称为多嘧啶结合蛋白(PTB)的蛋白质，它在决定哪种选择性剪接模式将发生方面发挥着重要作用。此外，它在某些mRNAs的解码过程中也起着重要作用。PTB是一种与RNA结合(相互作用)的蛋白质，表现出对特定RNA序列的偏好。PTB实际上有四个不同的RNA结合面，因此一个PTB分子可以同时与四个不同的RNA片段相互作用。这种多位点结合被认为是PTB如何调节剪接模式并促进某些mRNAs解码的关键。我们已经开发了一些方法来确定PTB与任何RNA的结合方向(即四个RNA结合面中的哪个结合到RNA上的哪个位点)，并找出这四个PTB-RNA相互作用中的哪一个对PTB的生物学作用至关重要。我们将使用这些方法来研究(I)PTB如何调控三个已知需要PTB结合才能启动解码的不同mRNAs的解码，以及(Ii)PTB如何调控三个不同的前mRNAs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.