Ribosome profiling of Trypanosoma brucei

布氏锥虫的核糖体分析

• 批准号: 8093893
• 负责人: Peter John Myler
• 金额: $ 28.51万
• 依托单位: SEATTLE BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8093893
• 关键词: African Trypanosomiasis; Bioinformatics; Cessation of life; Chagas Disease; Code; Complex; Couples; Cutaneous; Data; Development; Disease; Drug Delivery Systems; Gene Expression; Generations; Genes; Genome; Human; Individual; Insecta; Knowledge; Leishmania; Measures; Messenger RNA; Morbidity - disease rate; Open Reading Frames; Parasites; Pharmaceutical Preparations; Phenotype; Play; Process; Proliferating; Protein Biosynthesis; Proteins; RNA library; Relative (related person); Resistance; Ribosomes; Role; Sampling; Staging; Technology; Time; Transcriptional Regulation; Translating; Translations; Trypanosoma brucei brucei; Trypanosoma cruzi; Visceral; Work; mortality; new technology; next generation; nuclease; pathogen; tool

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. PROJECT NARRATIVE: The parasite Trypanosoma brucei causes fatal human African trypanosomiasis (sleeping sickness) and drugs to treat the disease are toxic and facing resistance. Generating new drugs requires knowledge of which proteins are expressed in the disease-causing stages of parasite development. This project will apply a new technology to measure the initial steps of protein synthesis for all genes in the infective as compared to non-infective stages, thereby providing new information on candidate drug targets.

描述（由申请人提供）：布氏锥虫是非洲锥虫病（“昏睡病”）的病原体，每年导致 50,000 多人死亡。相关的锥虫病原体，包括克氏锥虫（恰加斯病的病原体）和众多利什曼原虫（引起多种内脏、皮肤粘膜和皮肤疾病），在全世界范围内导致更高的发病率和死亡率。这些寄生虫中的每一种都经历了复杂的发育周期，在哺乳动物和昆虫宿主之间交替，以及增殖和非增殖阶段。锥虫基因表达究竟如何在每个阶段产生不同的表型目前尚不清楚，但基因特异性转录控制的相对贡献较低。转录后 mRNA 加工和稳定性的差异无疑起着重要作用，但寄生虫发育过程中 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，结合分离核糖体占据的 mRNA 的特定“足迹”的能力（这是翻译）与下一代测序的深度和广度。目标 1 将利用易于培养的非病原性昆虫阶段形式，在 T. brucei 中建立核糖体保护技术。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 样本中生成无偏差文库以进行下一代测序。它还将包括生物信息学管道的成熟，以分析所得序列数据。目标 2 将扩展到寄生虫的致病、哺乳动物阶段，并鉴定与非感染性形式相比，在感染性布氏锥虫发育过程中在翻译水平上受到调节的基因。拟议的工作将为研究锥虫基因表达提供一个重要的新工具，全面了解布氏锥虫中翻译控制的作用及其机制的线索，以及有关单个基因产物翻译程度的新信息，例如潜在的药物靶点。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，并且有潜力识别非规范蛋白质编码开放阅读框，从而显着增强正在进行的基因组注解。 项目叙述：寄生虫布氏锥虫会导致致命的人类非洲锥虫病（昏睡病），而治疗该疾病的药物有毒且面临耐药性。生产新药需要了解哪些蛋白质在寄生虫发育的致病阶段表达。该项目将应用新技术来测量感染阶段与非感染阶段所有基因的蛋白质合成的初始步骤，从而提供有关候选药物靶点的新信息。