Ribosome profiling of Trypanosoma brucei

描述（由申请人提供）：布鲁氏锥虫是非洲锥虫病（“昏睡病”）的病原体，每年造成5万多人死亡。相关的锥虫病病原体，包括克氏锥虫（恰加斯病的病原体）和许多利什曼原虫物种（引起各种内脏、粘膜和皮肤疾病），在世界范围内造成更高的发病率和死亡率。这些寄生虫中的每一种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，以及增殖和非增殖阶段。目前还不清楚锥虫基因表达如何在每个阶段产生不同的表型，但基因特异性转录控制的相对贡献很低。mRNA转录后加工和稳定性的差异无疑发挥了重要作用，但在寄生虫发育过程中，mRNA和蛋白质丰度之间的相关性较差，表明翻译和/或翻译后控制也很重要。该项目旨在通过应用最近描述的技术，将分离被核糖体（翻译指标）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，在任何特定时间内，全球和定量地评估每个mRNA被积极翻译的速率。目的1将利用易培养的非致病性昆虫阶段形式，建立布鲁氏体核糖体保护技术。它将优化核酸酶处理的条件，以保存核糖体保护的mRNA片段，并从RNA样本中生成无偏文库，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。目标2将扩展到寄生虫的致病性，哺乳动物阶段，并确定在感染与非感染形式的布氏体发育过程中在翻译水平上受调节的基因。这项工作将为研究锥虫基因表达提供一个重要的新工具，对布鲁氏锥虫翻译控制的作用和机制有一个全面的认识，并对单个基因产物的翻译程度，如潜在的药物靶点，提供新的信息。此外，它应该解决目前关于许多最近发现的仅包含短开放阅读框的rna的功能的争论，并有可能识别非规范的蛋白质编码开放阅读框，从而显著增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.