Regulation of mRNA Turnover in Trypanosomes

描述（由申请人提供）：锥虫病和利什曼病是世界上发展中国家的主要疾病。在这些地区，目前有10万人感染锥虫。人类疾病包括非洲锥虫病（“昏睡病”）、恰加斯病（南美和中美洲地区流行）以及皮肤、粘膜和内脏利什曼病（中东和印度次大陆部分地区流行）。非洲锥虫病通过携带布鲁氏锥虫亚种的采采蝇的叮咬传播给人类。据世界卫生组织估计，疾病负担为205万残疾调整生命年。治疗费用很高，未经治疗的感染通常会导致死亡。然而，锥虫之间共享许多代谢途径，这些途径与人类途径有足够的不同，可以用于开发比目前可用的治疗方法更有效的治疗方法。观察到锥虫虫在其人类和昆虫宿主中以不同的形式出现，提出了一种可能的治疗方法。这些在生命周期不同阶段的不同形式可能反映了mRNA稳定性的差异。本研究的目的是了解mrna在锥虫体内是如何代谢的。在这些寄生虫中，mRNA稳态水平的转录调控似乎很少。因此，在一个生命周期形式中，不同mRNA之间存在的mRNA稳定性差异可能部分受到mRNA衰变差异的调节。不同生命周期形式中mRNA表达模式的差异也可能部分由mRNA转换的差异控制。我们提出鉴定和表征两个重要的mRNA衰变的关键酶活性。我们的目标是：(1)纯化和克隆锥虫的脱帽酶；(2)纯化并克隆锥虫死烯化核酸酶；(3)评估脱帽酶和去烯酶在体内mRNA转换中的作用。我们最近开发了一种无细胞系统，能够胜任这两种酶的活性。这项工作是我们提出的实验计划的关键的第一步。

DESCRIPTION (provided by applicant): Trypanosomiasis and Leishmaniasis are major diseases in developing countries throughout the world. In these areas 100,000 people are currently infected with trypanosomes. Human illness includes African trypanosomiasis ("sleeping sickness"), Chagas' disease (endemic in regions of South and Central America) and cutaneous, mucocutaneous and visceral Leishmaniasis (endemic in parts of the Middle East and the Indian subcontinent). African trypanosomiasis is spread to humans by the bite of tsetse flies that harbor the Trypanosoma brucei subspecies. The disease burden is estimated by the World Health Organization to be 2.05 million Disability Adjusted Life Years. The cost of treatment is high, and untreated infection usually results in death. However, trypanosomatids share many metabolic pathways among themselves that differ enough from human pathways to be exploited in the development of therapeutics that are more efficacious than those currently available. One possible therapeutic approach is suggested by the observation that trypanosomatids occur in different forms in their human and insect hosts. These differing forms at various stages of the life cycle may reflect differences in mRNA stability. The purpose of this proposal is to gain an understanding of how mRNAs are metabolized in trypanosomes. There appears to be little transcriptional regulation of mRNA steady state levels in these parasites. Therefore, the differences in mRNA stability that exist among different mRNAs in one life cycle form may be regulated, in part, by differential mRNA decay. Differences in mRNA expression patterns among the different life cycle forms is also likely to be controlled, in part, by differential mRNA turnover. We propose to identify and characterize two crucial enzymatic activities important in mRNA decay. Our aims are (1) to purify and clone the decapping enzyme from trypanosomes; (2) to purify and clone the deadenylating nuclease from trypanosomes; and (3) to assess the roles of the decapping enzyme and deadenylase in mRNA turnover in vivo. We have recently developed a cell-free system that is competent for both of these enzymatic activities. This work is a key first step in our proposed experimental plan.