Characterization of the Role of PSF During pre-mRNA Splicing

PSF在pre-mRNA剪接中的作用。PTB相关剪接因子（PSF）是一种100kD RNA结合蛋白，最初是通过与多聚嘧啶束结合蛋白（PTB或hnRNP I）共同纯化而确定的。剪接提取物中PSF的缺失导致剪接第二步受阻，这可以通过添加重组PSF来挽救，这表明PSF是必不可少的第二步剪接因子。为了进一步表征PSF，进行了一系列实验来分析其RNA结合特异性并确定蛋白-蛋白相互作用伙伴。初步结果表明，PSF与U5 snRNA （stem 1b）中一个系统发育保守的系统结构结合。该项目的目标之一是确定PSF对包含一条或两条链1b的rna的亲和力，然后直接分析PSF-U5相互作用并绘制PSF在U5上的确切结合位点。从突变分析来看，stem 1b的序列不仅对系统本身的形成很重要，而且作为特定蛋白质的结合位点也很重要。这些结果表明，PSF与U5的茎1b结合，PSF-U5相互作用的破坏导致剪切的第二步受阻。为了直接验证这一假设，将使用荧光光谱进行一系列定量PSF-U5 RNA结合试验，然后进行体外功能试验，其中U5 snRNA将从剪接提取物中去除，然后用野生型或突变的U5 snRNA重建。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。这些实验将通过建立细胞系进行体内分析来补充，在细胞系中，PSF的两个等位基因都被破坏并被诱导的PSF转基因所取代。因此，所有破坏PSF结合的U5snRNA突变都将在体外和体内测试其对剪接的影响。大多数高级真核生物基因的表达需要有效和准确地切除中间序列（内含子），以便翻译功能蛋白。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种蛋白质和蛋白质- rna复合物的大复合体。剪接体组分中最具特征的是小核rna (snrna) U1、U2、U4、U5和U6，它们在剪接体的组装和催化中都起着核心作用。尽管已经确定了几个因素，特别是剪接第一步所需的那些因素，但对剪接体的蛋白质成分知之甚少。相比之下，第二步剪接所需的蛋白质的鉴定和功能表征相当滞后，特别是在高等真核生物中。该项目的主要目标是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.