Assembly and stability of supramolecular PTB: exon complexes

描述(申请人提供)：选择性剪接是产生多种蛋白质异构体的主要机制。对选择性剪接的调控通常需要特定于细胞类型或发育阶段的蛋白质，但这种多样性阻碍了对这一过程的分子机制的定义。在两个神经元前mRNAs的剪接中，一种蛋白质的功能、生化和生物物理性质为描述机制提供了机会。 我们的目标是了解多嘧啶结合蛋白(PTB)对c-src N1外显子和GABAA受体2神经元外显子排斥的机制。已知PTB在非神经细胞中是排除c-src前-mRNA的N1外显子的必要条件和充分条件，也是在非神经细胞中排除GABAA受体？2前-mRNA的神经元外显子所必需的。PTB结合在这些RNA的3‘剪接位点附近，但它的结合如何有效地隔离剪接位点尚不清楚。同样令人费解的是这种抑制被其他蛋白质解除的机制。 具体目标1致力于鉴定在大鼠GABA前-mRNA内含子/外显子上形成的PTB复合体。特定目的2详细说明PTB与小鼠c-src N1前mRNA外显子和侧翼内含子的相互作用。我们使用凝胶迁移率改变分析、硝酸纤维滤膜结合、酶足迹、荧光各向异性和核磁共振来确定结合亲和力、化学计量比、PTB与RNA结合的RNA位点以及PTB与RNA接触的结构域，以及荧光波动光谱来观察荧光标记的PTB分子的交换。 我们的数据导致了可检验的假设，即PTB的两半具有专门的RNA结合位点。两个N-末端结构域识别结构RNA中的多嘧啶序列，而两个C-末端结构域结合非结构RNA；这些RNA位点可以位于相同或不同的RNA上。在报道的拮抗剂nPTB存在的情况下，将测量PTB与复合体的结合/解离，以表征其抑制解除的机制。

DESCRIPTION (provided by applicant): Alternative splicing is the primary mechanism for generation of multiple protein isoforms. Regulation of alternative splicing often requires proteins specific to a cell type or developmental stage, but this very diversity has prevented definition of a molecular mechanism of the process. The functional biochemical and biophysical properties of one protein in the splicing of two neuronal pre-mRNAs offers the opportunity to describe a mechanism. Our goal is to understand the mechanism of exon exclusion of the c-src N1 exon and the GABAA receptor ?2 neuron exon by the polypyrimidine tract binding protein (PTB). PTB is known to be necessary and sufficient for exon exclusion of the N1 exon of c-src pre-mRNA in non-neural cells, and is required for exclusion of the neuron exon of the GABAA receptor ?2 pre-mRNA in non-neural cells. PTB binds near the 3' splice sites of these RNA, but how its binding effectively sequesters the splice site is obscure. Equally obscure is the mechanism by which this repression is relieved by other proteins. Specific Aim 1 is devoted to the characterization of PTB complexes formed on the rat GABA pre-mRNA intron/exon. Specific Aim 2 details interactions of PTB with mouse c-src N1 pre-mRNA exon and flanking introns. We use gel mobility shift assays, nitrocellulose filter binding, enzymatic footprinting, fluorescence anisotropy, and NMR to determine binding affinity, stoichiometry, the RNA sites bound by PTB, and the domains of PTB in contact with the RNA, and fluorescence fluctuation spectroscopy to observe the exchange of fluorescently labeled PTB molecules. Our data have led to the testable hypothesis that the two halves of PTB have specialized RNA binding sites. The two N-terminal domains recognize polypyrimidine tracts in structured RNAs, while the two C-terminal domains bind unstructured RNAs; these RNA sites could be on the same or different RNAs. PTB association/dissociation from complexes will be measured in the presence of nPTB, a reported antagonist, to characterize its mechanism of repression relief.