Ribosome profiling of Trypanosoma brucei

描述(申请人提供)：布鲁氏锥虫是非洲锥虫病(“昏睡病”)的病原体，每年造成50,000多人死亡。与锥虫相关的病原体，包括克氏锥虫(恰加斯病的病原体)和众多利什曼原虫(导致各种内脏、黏膜皮肤和皮肤病)，在世界范围内造成更高的发病率和死亡率。每种寄生虫都经历了一个复杂的发育周期，在哺乳动物和昆虫宿主之间交替，以及增殖期和非增殖期。锥虫基因表达如何在每个阶段产生不同的表型目前尚不清楚，但基因特异性转录调控的相对贡献很低。转录后mRNA的加工和稳定性的差异无疑是主要原因，但在寄生虫发育过程中，mRNA和蛋白质丰度之间的相关性很差，这表明翻译和/或翻译后控制也很重要。该项目寻求通过应用一种最近描述的技术，将分离核糖体(翻译指标)所占据的mRNA的特定“足迹”的能力与下一代测序的深度和广度结合起来，以全局和定量地评估在任何特定时间内每一个mRNA被积极翻译的速率。目的1利用易于培养的非致病昆虫阶段形态，建立布鲁氏毛滴虫核糖体保护技术。它将优化核酸酶处理的条件，以保存受核糖体保护的mRNA片段，并从RNA样本中生成无偏倚的文库，用于下一代测序。它还将包括成熟的生物信息学管道，以分析产生的序列数据。目的2将扩展到寄生虫的致病哺乳动物阶段，并与非感染形式相比，确定在布鲁氏毛滴虫发育过程中在翻译水平上受调控的基因。这项拟议的工作将为研究锥虫的基因表达提供一个重要的新工具，从而全面了解翻译控制在布氏锥虫中的作用及其机制的线索，以及关于单个基因产物的翻译程度的新信息，例如潜在的药物靶标。此外，它应该解决目前关于许多最近发现的RNA的功能的争论，这些RNA只包含较短的开放阅读框架，并有可能识别非规范的蛋白质编码开放阅读框架，从而显著加强正在进行的基因组注释。

DESCRIPTION (provided by applicant): Trypanosoma brucei, the causative agent of African trypanosomiasis ("sleeping sickness"), causes more than 50,000 deaths annually. Related trypanosomatid pathogens, including Trypanosoma cruzi (the causative agent of Chagas' disease) and numerous Leishmania species (which cause a diverse spectrum of visceral, mucocutaneous, and cutaneous disease), cause even more morbidity and mortality worldwide. Each of these parasites undergoes a complex developmental cycle, alternating between mammalian and insect hosts, as well as proliferating and non-proliferating stages. Exactly how trypanosomatid gene expression gives rise to the different phenotypes at each stage is currently not well understood, but the relative contribution of gene-specific transcriptional control is low. Differences in post-transcriptional mRNA processing and stability undoubtedly play major roles but the poor correlation between mRNA and protein abundance during parasite development indicates that translational and/or post-translational controls are also important. This project seeks to globally and quantitatively assess the rate at which each mRNA is actively translated at any particular time by applying a recently-described technology that couples the ability to isolate the specific "footprints" of mRNAs that are occupied by ribosomes (an indicator of translation) with the depth and breadth of next generation sequencing. Aim 1 will establish the ribosome protection technology in T. brucei, using readily cultured non-pathogenic insect stage forms. It will optimize conditions for nuclease treatment to preserve mRNA fragments protected by ribosomes and for the generation of unbiased libraries from the RNA samples for next generation sequencing. It will also include maturation of the bioinformatics pipeline to analyze resulting sequence data. Aim 2 will expand into the pathogenic, mammalian stages of the parasite, and identify genes that are regulated at the level of translation during T. brucei development in infective as compared to non-infective forms. The proposed work will provide an important new tool for studying trypanosomatid gene expression, yielding a comprehensive view of the role of translational control in T. brucei and clues to it mechanisms, as well as new information on the extent of translation of individual gene products, such as potential drug targets. In addition, it should resolve the current debate over the function of the numerous recently identified RNAs that contain only short open-reading frames, and has the potential to identify non-canonical protein-coding open-reading frames, thus significantly enhancing the ongoing genome annotation.