Functional and structural characterization of spliceosomal cyclophilins

描述（由申请人提供）：剪接体是RNA和蛋白质的复杂和动态集合，其从前体mRNA转录物中去除内含子。剪接机制的改变与从癌症到色素性视网膜炎等多种人类疾病有关。深入了解剪接导致这些病理状态的机制需要了解剪接体内各个成分的功能。在这一建议中，计划阐明亲环素类肽脯氨酰异构酶在剪接中的作用。亲环素是一种高度保守的蛋白质，也是环孢菌素药物的靶点，但其生理功能仍是个谜。作为结构基因组学计划的一部分，我已经将几种亲环素的结构解析到原子分辨率，并产生了大量可溶性蛋白质表达构建体。我还根据它们在溶液中的催化活性表征了这些蛋白质，并基于计算机模拟假设了潜在的底物特异性。然而，这种酶的体内底物没有定义，使得我以前的结果难以验证。基于几种核亲环素在纯化的人剪接复合物中富集的发现，剪接体亲环素的靶点可能会提供大量关于亲环素：底物特异性的信息。此外，在剪接体机制中发现的亲环蛋白家族成员的绝对数量及其在整个剪接复合物中的独特分布表明这些蛋白质可能对适当的剪接活性至关重要。为了检验这一假设，我将首先接受培训，使用Jurica实验室优化的体外剪接测定法，用重组形式的潜在剪接因子重建剪接体。我已经开始使用这种测定来测试在亲环蛋白PPIE存在下的剪接活性，并且可以表明这种蛋白质确实是正确剪接功能所必需的。接下来，我将在Jurica实验室接受后续研究的培训，这些研究旨在找到PPIE发挥作用的剪接阶段，并学习如何纯化剪接体复合物以用于质谱分析。这些研究将提供第一个洞察到个人剪接体相关亲环素在前mRNA剪接的功能重要性，并揭示剪接体组装的阶段，他们的目标。在掌握这些技术后，我将在自己的实验室中继续进行剪接体亲环素的研究工作，在那里我将分离出与亲环素相关的剪接体的单个组分。然后，我将对亲环蛋白和感兴趣的蛋白质进行生物物理分析，并利用X射线晶体学解决这些蛋白质复合物的复杂结构。这些剪接体亲环素的结构/功能研究的结果将扩大我们的理解剪接机制，包括亲环素特异性蛋白质的作用：蛋白质相互作用和脯氨酸异构化剪接体内。 公共卫生相关性：剪接机制的改变与从癌症到色素性视网膜炎的各种人类疾病有关。深入了解剪接导致这些病理状态的机制需要了解剪接体内各个成分的功能。这项工作将作为一个平台，开发小分子试剂或蛋白质突变体，专门针对关键剪接体组件。这些试剂将用于阐明剪接体参与疾病病理学。

DESCRIPTION (provided by applicant): The spliceosome is a complex and dynamic collection of RNA and proteins that removes introns from precursor mRNA transcripts. Alterations in the splicing machinery are associated with a diverse set of human diseases, ranging from cancer to retinitis pigmentosa. Insight into the mechanisms by which splicing leads to these pathological states requires an understanding of the functions of individual components within the spliceosome. In this proposal are plans to elucidate the role of the cyclophilin class of peptdyl-prolyl isomerases in splicing. Cyclophilins are highly conserved proteins and the target of the drug cyclosporin but their physiological functions remain enigmatic. I have solved the structures of several cyclophilins to atomic resolution as part of a structural genomics initiative and generated large numbers of soluble protein expression constructs. I also characterized these proteins in terms of their catalytic activities in solution and hypothesized potential substrate specificity based on in silico modeling. However, in vivo substrates for this enzyme class are not defined, making validation of my previous results difficult. Based on the finding that several nuclear cyclophilins are enriched in purified human splicing complexes, it is likely that the targets of spliceosomal cyclophilins will provide a great deal of information concerning cyclophilin:substrate specificity. Additionally, the sheer number of the cyclophilin family members found within the spliceosomal machinery and their unique distribution throughout splicing complexes indicate that these proteins are likely to be crucial for proper splicing activity. In order to test this hypothesis I will first be trained in the use of an in vitro splicing assay optimized in the Jurica lab to reconstitute spliceosomes with recombinant versions of potential splicing factors. I have already begun to use this assay to test for splicing activity in the presence of the cyclophilin PPIE, and can show that indeed this protein is necessary for proper splicing function. Next I will be trained in the Jurica lab in follow-up studies designed to find the stage of splicing at which PPIE exerts its effect, and learn how to purify spliceosomal complexes for use in mass spectrometric analysis. These studies will provide the first insight into the functional importance of the individual spliceosome-associated cyclophilins in pre-mRNA splicing and reveal the stage of spliceosome assembly that they target. After mastering these techniques I will carry on my work in spliceosomal cyclophilins in my own lab, where I will isolate individual components of the spliceosome found to associate with cyclophilins. I will then perform biophysical assays on the cyclophilin and protein of interest, and direct my efforts to solving complex structures of these protein complexes utilizing x-ray crystallography. The results of these structure/function studies of spliceosomal cyclophilins will expand our understanding of splicing mechanism to include the roles of cyclophilin specific protein:protein interactions and proline isomerization within the spliceosome. PUBLIC HEALTH RELEVANCE: Alterations in the splicing machinery are associated with a diverse set of human diseases ranging from cancer to retinitis pigmentosa. Insight into the mechanisms by which splicing leads to these pathological states requires an understanding of the functions of individual components within the spliceosome. This work will serve as a platform for developing small molecule reagents or protein mutants that specifically target key spliceosome components. These reagents will be used to elucidate the spliceosome's involvement in disease pathologies.