Functional and structural characterization of spliceosomal cyclophilins

描述（由申请人提供）：剪接体是一种复杂和动态的RNA和蛋白质集合，可以从前体mRNA转录物中去除内含子。剪接机制的改变与多种人类疾病有关，从癌症到色素性视网膜炎。要深入了解剪接导致这些病理状态的机制，需要了解剪接体中各个成分的功能。在这个建议是计划阐明亲环类肽-脯氨酸异构酶在剪接中的作用。亲环蛋白是一种高度保守的蛋白，是环孢素类药物的靶点，但其生理功能仍是一个谜。作为结构基因组学计划的一部分，我已经解决了几个亲环蛋白的原子分辨率结构，并生成了大量的可溶性蛋白表达结构。我还根据这些蛋白质在溶液中的催化活性对其进行了表征，并基于硅模型假设了潜在的底物特异性。然而，这类酶的体内底物尚未定义，这使得验证我以前的结果变得困难。基于在纯化的人类剪接复合体中富集了几种核亲环蛋白的发现，剪接体亲环蛋白的靶标可能会提供大量关于亲环蛋白底物特异性的信息。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剪接复合体中的独特分布表明，这些蛋白可能对适当的剪接活性至关重要。为了验证这一假设，我将首先接受使用Jurica实验室优化的体外剪接试验的培训，用潜在剪接因子的重组版本重建剪接体。我已经开始使用这种方法来测试亲环蛋白PPIE存在下的剪接活性，并且可以证明这种蛋白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，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。这些研究将首次深入了解单个剪接体相关亲环蛋白在mrna前剪接中的功能重要性，并揭示它们所针对的剪接体组装阶段。在掌握了这些技术之后，我将在自己的实验室中继续我在剪接体亲环蛋白方面的工作，在那里我将分离出与亲环蛋白相关的剪接体的单个成分。然后，我将对感兴趣的亲环蛋白和蛋白质进行生物物理分析，并利用x射线晶体学指导我的工作来解决这些蛋白质复合物的复杂结构。这些剪接体亲环蛋白的结构/功能研究结果将扩展我们对剪接机制的理解，包括亲环蛋白特异性蛋白的作用：剪接体内的蛋白质相互作用和脯氨酸异构化。

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.