Assembly and stability of supramolecular PTB: exon complexes

超分子PTB的组装和稳定性：外显子复合物

基本信息

批准号：
7925986
负责人：
KATHLEEN B HALL
金额：
$ 30.9万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7925986
关键词：
3&apos Splice Site Affinity Alternative Splicing Binding Binding Sites Biochemical Biological Models C-terminal Cell Nucleus Cells Chemicals Complex Data Development Dissociation Equilibrium Exclusion Exons Fluorescence Fluorescence Anisotropy Fluorescence Spectroscopy Generations Goals In Vitro Introns Label Length Location Maps Measures Messenger RNA Methods Modeling Molecular Molecular Conformation Mus N-terminal Neurons Nucleotides Polypyrimidine Tract-Binding Protein Process Property Protein Binding Protein Isoforms Proteins Pyroxylin RNA RNA Binding RNA Splicing RNA-Binding Proteins Rattus Regulation Relative (related person)Reporting Repression Role SRC gene Site Spectrum Analysis Staging Structure Tertiary Protein Structure cell type gamma-Aminobutyric Acid gel mobility shift assay genetic regulatory protein mRNA Precursor mutant preference prevent protein complex protein protein interaction receptor research study stoichiometry

项目摘要

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.

描述（由申请人提供）：替代剪接是生成多种蛋白质同工型的主要机制。对替代剪接的调节通常需要特定于细胞类型或发育阶段的蛋白质，但是这种多样性阻止了该过程的分子机制的定义。一种蛋白质在两个神经元前MRNA的剪接中的功能性生化和生物物理特性提供了描述一种机制的机会。我们的目标是了解外显子排除C-SRC N1外显子和GABAA受体？2神经元外显子，而息肉酰胺类结合蛋白（PTB）。已知PTB在非神经细胞中的C-SRC前MRNA的N1外显子排除外显子是必不可少的，并且是排除非神经细胞中GABAA受体的神经元外显子所必需的。 PTB在这些RNA的3'剪接位点附近结合，但是其结合方式如何有效地隔离剪接位点是晦涩的。同样晦涩的是这种抑制作用被其他蛋白质缓解的机制。具体目标1致力于在大鼠GABA前MRNA内含子/外显子上形成的PTB复合物的表征。特定的目标2细节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转移测定，硝酸纤维素过滤器结合，酶足迹，荧光各向异性和NMR来确定结合亲和力，化学计量，pTB结合的RNA位点，PTB结合的RNA位点以及与RNA和荧光频率相关的PTB域，PTB的结构范围。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末端结构域在结构化RNA中识别息肉酰胺域，而两个C末端结构域结合了非结构化的RNA。这些RNA位点可以在相同或不同的RNA上。将在据报道的拮抗剂NPTB的存在下测量与复合物的PTB关联/解离，以表征其抑制作用的机理。