Functional and structural characterization of spliceosomal cyclophilins

项目总结 剪接体是一个复杂的、动态的RNA和蛋白质的集合，它从 前体mRNA转录本。剪接机械的改变与一组不同的人类 疾病，从癌症到视网膜色素变性。洞察剪接导致 这些病理状态需要了解各个组件在 剪接体。在这项提案中，计划阐明亲环素类肽基-脯氨基的作用 剪接中的异构酶。 亲环素是高度保守的蛋白质，也是药物环孢素的靶点，但其生理功能 功能仍然是个谜。我已经解决了几个亲环素的结构到原子分辨率，作为 结构基因组学倡议并产生了大量的可溶性蛋白表达构建体。我也是 根据它们在溶液中的催化活性和假设的潜在底物来表征这些蛋白质 基于电子计算机建模的特异性。然而，该酶类的体内底物没有定义， 使得验证我之前的结果变得困难。基于这一发现，几种核亲环素是 富含纯化的人类剪接复合体，很可能是剪接体亲环素的靶标 提供大量有关亲环素的信息：底物专一性。此外，纯粹的数字 在剪接体机制中发现的亲环素家族成员及其独特的分布 贯穿剪接的复合体表明，这些蛋白质对于正确的剪接活性可能是至关重要的。 为了验证这一假设，我将首先接受使用体外剪接试验的培训，该体外剪接试验在 Jurica实验室用潜在剪接因子的重组版本重建剪接体。我已经去过了 开始使用这种方法测试在亲环素PPIE存在的情况下的剪接活性，并可以表明 事实上，这种蛋白质是正常剪接功能所必需的。接下来，我将在尤里卡实验室接受后续培训 旨在找到PPIE发挥作用的剪接阶段，并学习如何净化的研究 用于质谱分析的剪接体复合体。这些研究将提供第一次洞察 单个剪接体相关的亲环素在前mRNA剪接中的功能重要性和 揭示了他们的目标剪接体组装的阶段。 在掌握了这些技术之后，我将在我自己的实验室继续我的剪接体亲环素工作，在那里我 将分离被发现与亲环素相关的剪接体的个别成分。然后我会表演 亲环素和感兴趣的蛋白质的生物物理分析，并指导我的努力来解决复杂的结构 利用X射线结晶学对这些蛋白质复合体进行分析。这些结构/功能研究的结果 剪接体亲环素将扩大我们对剪接机制的理解，包括 亲环素特异性蛋白：剪接体内的蛋白质相互作用和脯氨酸异构化。

PROJECT SUMMARY 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.