Characterization of the Role of PSF During pre-mRNA Splicing

Patton, James G.NSF MCB-9974542摘要表征 PSF 在前 mRNA 剪接过程中的作用。 PTB 相关剪接因子 (PSF) 是一种 100kD RNA 结合蛋白，最初是通过与聚嘧啶束结合蛋白（PTB 或 hnRNP I）共纯化而鉴定的。 剪接提取物中 PSF 的耗尽会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 是重要的第二步剪接因子。 为了进一步表征 PSF，进行了一系列实验来分析其 RNA 结合特异性并鉴定蛋白质-蛋白质相互作用伙伴。 初步结果表明，PSF 与 U5 snRNA（茎 1b）内系统发育保守的茎结构结合。 该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-U5 相互作用并绘制 PSF 在 U5 上的精确结合位点。 从突变分析来看，茎 1b 的序列似乎不仅对于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。 这些结果表明 PSF 与 U5 的茎 1b 结合，并且 PSF-U5 相互作用的破坏导致剪接的第二步受阻。 为了直接检验这一假设，将使用荧光光谱法进行一系列定量 PSF-U5 RNA 结合测定，然后进行功能性体外测定，其中 U5 snRNA 将从剪接提取物中耗尽，然后用野生型或突变型 U5 snRNA 进行重建。这将使我们能够测试特定 U5 突变对剪接和 PSF 结合的影响。 这些实验将通过创建细胞系后的体内分析来补充，其中PSF的两个等位基因都被破坏并被诱导型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其对剪接的影响。 大多数高等真核基因的表达需要有效且准确地切除间插序列（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 复合物的大型复合物。 剪接体组分中特征最明显的是小核 RNA (snRNA) U1、U2、U4、U5 和 U6，它们在剪接体组装和催化中发挥核心作用。 尽管已经鉴定了几个因素，特别是剪接第一步所需的因素，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却大大滞后，特别是在高等真核生物中。 该项目的总体目标是增加对人类剪接第二步的了解，并确定 PSF 与 U5 snRNA 的关联是否对该过程至关重要。

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.