Characterization of the Role of PSF During pre-mRNA Splicing

PSF在前信使核糖核酸剪接中作用的特征。PTB相关剪接因子(PSF)是一种100kD的RNA结合蛋白，最初是通过与多嘧啶结合蛋白(PTB或hnRNP I)的共纯化而被鉴定出来的。从剪接提取物中耗尽PSF会阻碍第二步剪接，而重组PSF的加入可以挽救这一障碍，这表明PSF是一个必不可少的第二步剪接因子。为了进一步表征PSF，人们进行了一系列实验来分析其RNA结合特异性，并确定蛋白质-蛋白质相互作用伙伴。初步结果表明，PSF与U5SnRNA(茎1b)中的系统发育保守的茎结构结合。该项目的目标之一是确定PSF与包含茎1b的一条或两条链的RNA的亲和力，然后直接分析PSF-U5相互作用并定位PSF在U5上的确切结合位点。从突变分析来看，茎1b的序列似乎不仅对茎本身的形成很重要，而且作为特定蛋白质的结合位点也很重要(S)。结果表明，PSF与U5的茎1b结合，PSF-U5相互作用的中断导致剪接的第二步受阻。为了直接验证这一假设，我们将使用荧光光谱进行一系列定量的PSF-U5 RNA结合分析，然后进行功能体外实验，其中U5 SnRNA将从剪接提取物中去除，然后与野生型或突变的U5 SnRNA重组。这将使我们能够测试特定的U5突变对剪接和PSF结合的影响。这些实验将得到体内分析的补充，在建立的细胞系中，PSF的两个等位基因都被破坏，并被可诱导的PSF转基因取代。因此，所有破坏PSF结合的U5SnRNA突变都将在体外和体内测试它们对剪接的影响。大多数高等真核基因的表达需要有效和准确地切除中间序列(内含子)以允许功能蛋白的翻译。内含子的去除在剪接体内分两步进行，剪接体是一个包含多个蛋白质和蛋白质-RNA复合体的大型复合体。剪接体成分中最具特点的是小核RNA(SnRNAs)U1、U2、U4、U5和U6，它们在剪接体组装和催化中都发挥着核心作用。尽管已经确定了几个因素，特别是剪接的第一步所需的因素，但Muchless对剪接体的蛋白质成分知之甚少。相比之下，剪接的第二步所需蛋白质的鉴定和功能特性却相当滞后，特别是在高等真核生物中。该项目的主要目标是寻求增加对人类剪接第二步的了解，并确定PSF与U5 SnRNA的关联是否对这一过程至关重要。

Patton, James G.NSF MCB-9974542AbstractCharacterization of the Role of PSF During pre-mRNA Splicing. PTB-Associated Splicing Factor (PSF) is a 100kD RNA binding proteinthat was originally identified based on its co-purification withPolypyrimidine Tract Binding Protein (PTB or hnRNP I). Depletion of PSFfrom splicing extracts causes a block to the second step of splicing whichcan be rescued by the addition of recombinant PSF suggesting that PSF is anessential second step splicing factor. To further characterize PSF, aseries of experiments have been performed to analyze its RNA bindingspecificity and identify protein-protein interaction partners. Preliminaryresults suggest that PSF binds to a phylogenetically conserved stemstructure within U5 snRNA (stem 1b). One of the goals of this project isto determine the affinity of PSF for RNAs encompassing one or both strandsof stem 1b followed by direct analysis of PSF-U5 interaction and mapping ofthe exact binding site for PSF on U5. From mutational analyses, it appearsthat the sequence of stem 1b is important not only for the formation of thestem itself, but also as a binding site for a specific protein(s). Theseresults suggest that PSF binds to stem 1b of U5 and that disruption ofPSF-U5 interaction results in a block to the second step of splicing. Todirectly test this hypothesis, a series of quantitative PSF-U5 RNA bindingassays will be performed using fluorescence spectroscopy followed by afunctional in vitro assay in which U5 snRNA will be depleted from splicingextracts and then reconstituted with either wild type or mutated U5 snRNAs.This will allow us to test the effects of specific U5 mutations on bothsplicing and PSF binding. These experiments will be complemented by invivo analyses following the creation of cell lines in which both alleles ofPSF are disrupted and replaced by an inducible PSF transgene. Thus, all U5snRNA mutations that disrupt PSF binding will be tested for their effectson splicing both in vitro and in vivo. Expression of most higher eukaryotic genes requires thatintervening sequences (introns) be efficiently and accurately excised toallow translation of functional proteins. Removal of introns occurs in twosteps within the spliceosome, a large complex containing multiple proteinand protein-RNA complexes. Among the best characterized of the spliceosomecomponents are the small nuclear RNAs (snRNAs) U1, U2, U4, U5, and U6,which play central roles both in spliceosome assembly and catalysis. Muchless is known about the protein components of the spliceosome althoughseveral factors have been identified, particularly those required for thefirst step of splicing. In contrast, the identification and functionalcharacterization of proteins required for the second step of splicing haslagged considerably, particularly in higher eukaryotes. The broad goals ofthis project seek to increase knowledge about the second step of splicingin humans and determine whether association of PSF with U5 snRNA isessential for this process.