Characterization of the Role of PSF During pre-mRNA Splicing

Patton，James G.NSF MCB-9974542 AbstractCharacteristics of the Role of PSF During the Pre-mRNA Splicing. PTB相关剪接因子（PSF）是一种分子量为100 kD的RNA结合蛋白，最初是通过与多聚嘧啶结合蛋白（PTB或hnRNP I）共纯化而鉴定的。 从剪接提取物中去除PSF会导致第二步剪接的阻断，这可以通过加入重组PSF来挽救，表明PSF是一种必需的第二步剪接因子。 为了进一步表征PSF，进行了一系列实验以分析其RNA结合特异性并鉴定蛋白质-蛋白质相互作用伙伴。 初步结果表明，PSF结合到U 5 snRNA内的一个遗传上保守的茎结构（茎1b）。 本项目的目标之一是确定PSF对包含茎1b的一条或两条链的RNA的亲和力，然后直接分析PSF与U 5的相互作用并绘制PSF在U 5上的确切结合位点。 突变分析表明，茎1b的序列不仅对茎本身的形成很重要，而且作为一个特定蛋白质的结合位点也很重要。 这些结果表明PSF与U 5的茎1b结合，并且PSF-U 5相互作用的破坏导致剪接第二步的阻断。 为了直接验证这一假设，我们将使用荧光光谱法进行一系列PSF-U 5 RNA结合的定量分析，然后进行功能性体外分析，其中将从剪接提取物中去除U 5 snRNA，然后用野生型或突变型U 5 snRNA重建，这将使我们能够测试特定U 5突变对剪接和PSF结合的影响。 这些实验将通过体内分析来补充，随后创建细胞系，其中两个等位基因ofPSF被破坏并被诱导型PSF转基因取代。 因此，所有破坏PSF结合的U 5snRNA突变都将在体外和体内测试其对剪接的影响。 大多数高等真核生物基因的表达需要插入序列（内含子）被有效和准确地切除，以允许功能蛋白的翻译。 剪接体是一个含有多种蛋白质和蛋白质-RNA复合物的大复合体，内含子的去除分两步进行。 其中最具特征的剪接体组分是小核RNA（snRNA）U1，U2，U4，U 5和U6，它们在剪接体组装和催化中起着核心作用。 尽管已经确定了几个因子，特别是剪接第一步所需的因子，但对剪接体的蛋白质组分知之甚少。 相反，剪接第二步所需的蛋白质的鉴定和功能表征已经相当滞后，特别是在高等真核生物中。 该项目的广泛目标是寻求增加对人类剪接第二步的了解，并确定PSF与U 5 snRNA的结合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.