Comparative Analysis of Editosome in Trypanosomatids

描述(申请人提供)：该项目的父母资助为R37 AI4102，正常和突变锥虫的线粒体DNA。这个项目建立在父母资助研究的基础上，该研究确定了布鲁氏锥虫编辑机制(编辑体)的一些组成部分的蛋白质和基因。亲本资助为这种生物提供了重要的生物学见解，对不同动质体种的编辑体的比较分析将为评估编辑体的组成和功能的生理和进化提供一个特殊的机会。这一分析不仅在识别预测算法未调用的基因方面具有实用价值，而且为了解动体寄生虫RNA编辑的分子机制提供了宝贵的额外洞察水平。在这项提案中，我们将确定编辑体的组成，确定人类关键的主要动体寄生虫：布氏锥虫、克鲁兹锥虫和大利什曼原虫。这将通过利用布鲁氏锥虫的信息快速鉴定另外两种主要动质体寄生虫的编辑体基因和蛋白质来实现。该项目将采用电子分析(已经在布氏毛滴虫中识别出许多候选基因)和利用亲和标签技术与串联质谱学相结合的直接分析相结合的方法。此外，由于现有数据表明锥虫和利什曼原虫之间存在显著的结构差异，因此将通过电子显微镜(EM)确定具有催化活性的编辑小体的形态。这项建议的总体目标是确定这三个物种编辑小体的保守和分化的结构和功能特征。这些分析将有助于理解编辑体及其组成部分的功能，也有助于开发对这三个物种都有效的药物。

DESCRIPTION (provided by applicant): The parent grant for this project is R37 AI4102, Mitochondrial DNA of Normal and mutant Trypanosomes. This project builds on studies in the parent grant that identified proteins and genes for some of the components of the editing machinery (editosome) in Trypanosoma brucei. The parent grant provides significant biological insights for this organism, comparative analysis of the editosome in different kinetoplastid species will provide an exceptional opportunity to assess the physiology and evolution of editosome composition and function. This analysis will not only have practical value in the identification of genes not called by predictive algorithms but provides an invaluable additional level of insight on the molecular mechanisms of RNA editing in kinetoplastid parasites. In this proposal we will establish the composition of the editosome, determine the critical major kinetoplastid parasites of humans; T. brucei, T. cruzi, and Leishmania major. This will be accomplished by exploiting the information from T. brucei for rapid identification of editosome genes and proteins in the other two major kinetoplastid parasites. This project will employ a combination of in silico analyses (which have already identified many candidate genes in T. brucei) and direct assays that exploit affinity tag technology combined with tandem mass spectroscopy. Furthermore, the morphology of catalytically active editosomes will be determined by electron microscopy (EM), since the available data indicate substantial structural differences between Trypanosoma and Leishmania species. The overall goal of this proposal is to identify conserved and divergent structural and functional features of the editosome in these three species. These analyses will be useful for understanding the function of the editosome and its components and also for development of drugs that could be effective for all three species.