Regulation of RNA Editing in Leishmania

描述（由申请人提供）：利什曼原虫是白蛉传播的致病性原生动物，影响热带和亚热带地区的数百万人。这些寄生虫似乎依赖于活跃的线粒体功能，包括在其整个生命周期中通过RNA编辑进行mRNA加工，从而使RNA编辑机制成为有希望的药物靶标。然而，对哺乳动物阶段（无鞭毛体）的线粒体功能（包括RNA编辑）知之甚少。这一知识空白将通过拟议的试点研究加以解决，该研究将利用杜氏利什曼原虫和墨西哥利什曼原虫亚马逊亚种的体外前鞭毛体-无鞭毛体分化系统。我们将测试的假设，至少有一些的kDNA编码的转录物在RNA编辑的水平发育调控。这一假设是基于我们早期的观察，即呼吸复合物I（NADH脱氢酶）的某些组分的RNA编辑在利什曼原虫的前鞭毛体（相当于昆虫阶段）中被抑制，这表明这些组分在生命周期的不同阶段是必需的。具体目标是：（1）确定两种致病物种中大环保守区的序列;（2）确定这些物种的前鞭毛体中编辑的mRNA的序列;（3）建立无鞭毛体的分批培养物，并在未编辑和编辑的mRNA比率和编辑模式方面与前鞭毛体进行比较;（4）使用可用的抗体比较两个阶段中RNA编辑机器的酶和辅助蛋白质组分的水平。这些研究将揭示利什曼原虫细胞生物学的鲜为人知的方面，具有发现新的调控机制和药物靶点的潜力。

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.