Regulation of RNA Editing in Leishmania

描述（由申请人提供）：利什曼原虫是一种由白蛉传播的致病性原生动物，影响着热带和亚热带地区数百万人。这些寄生虫似乎依赖于活跃的线粒体功能，包括通过RNA编辑处理mRNA，从而使RNA编辑机制成为一个有希望的药物靶点。然而，在哺乳动物阶段（无纺体），线粒体的功能，包括RNA编辑，我们所知甚少。这方面的知识差距将通过拟议的试点研究来解决，该研究将利用多诺瓦利什曼原虫和墨西哥亚马逊利什曼原虫的体外原鞭毛体-无鞭毛体分化系统。我们将验证至少一些kdna编码转录本在RNA编辑水平上受到发育调节的假设。这一假设是基于我们早期的观察，即呼吸道复合体I （NADH脱氢酶）的某些成分的RNA编辑在利什曼原虫的螺旋体（相当于昆虫阶段）中是不必要的，这表明这些成分在生命周期的不同阶段是必需的。具体目标是：(1)确定两种病原菌最大环的保守区序列；(2)确定这些物种的promastigotes中编辑的mrna序列；(3)建立无性无性体的批量培养，并与原无性体进行未编辑和编辑mRNA比例和编辑模式的比较；(4)使用可用的抗体比较两个阶段RNA编辑机制的酶和辅助蛋白成分的水平。这些研究将阐明利什曼原虫细胞生物学鲜为人知的方面，并有可能发现新的调节机制和药物靶点。

DESCRIPTION (provided by applicant): Leishmanias are sandfly-transmitted pathogenic protozoa affecting millions of people in tropical and subtropical regions. These parasites appear to depend on active mitochondrial functions including mRNA processing by RNA editing, throughout their life cycle, thus making the RNA editing machinery a promising drug target. Little is known, however, about mitochondrial functions including RNA editing during the mammalian stage (amastigotes). This gap in knowledge will be addressed by the proposed pilot studies which will utilize in vitro promastigote-amastigote differentiation systems of Leishmania donovani and Leishmania mexicana amazonensis. We will test the hypothesis that at least some of the kDNA-encoded transcripts are developmentally regulated at the level of RNA editing. This hypothesis is based on our early observation that RNA editing for some components of respiratory Complex I (NADH dehydrogenase) is dispensable in promastigotes of Leishmania tarentolae (equivalent to insect stage) suggesting that these components are required at a different stage of the life cycle. The specific goals are: (1) to determine the sequences of the conserved region of the maxicircles in the two pathogenic species; (2) to determine the sequences of edited mRNAs in promastigotes of these species; (3) to establish batch cultures of axenic amastigotes and perform a comparison with promastigotes in respect to unedited and edited mRNA ratios and patterns of editing; (4) to compare levels of the enzymatic and auxiliary protein components of the RNA editing machinery at both stages using available antibodies. These studies will shed light on the poorly known aspects of Leishmania cell biology with a potential of discovery of novel regulatory mechanisms and drug targets.