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）：非洲锥虫病的病因（“睡眠疾病”），每年造成50,000多人死亡。相关的锥形病原体，包括克鲁氏锥虫（Cruzi（Chagas）疾病的病原体）和许多利什曼原虫（Leishmania）物种（引起多种内脏，粘膜皮肤病和皮肤病），导致全世界的发病率和死亡率更高。这些寄生虫中的每一个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。确切地说，当前尚不清楚锥虫基因表达如何引起每个阶段的不同表型，但是基因特异性转录控制的相对贡献很低。转录后mRNA加工和稳定性的差异无疑起着主要作用，但是在寄生虫发育过程中mRNA和蛋白质丰度之间的相关性差表明翻译和/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在任何特定时间内积极地翻译每个mRNA的速率，以隔离核糖体（翻译指标）与下一代序列的深度和广度来隔离MRNA的特定“足迹”，从而在任何特定时间内积极地翻译了每个mRNA。 AIM 1将使用容易培养的非致病昆虫阶段形式在T. Brucei中建立核糖体保护技术。它将优化核酸酶处理的条件，以保留受核糖体保护的mRNA片段，并从RNA样品中生成无偏的文库进行下一代测序。它还将包括生物信息学管道的成熟，以分析结果序列数据。 AIM 2将扩展到寄生虫的致病性哺乳动物阶段，并鉴定出在传染性形式相比，在传染性形式的布鲁氏乳杆菌发育过程中受调节的基因。拟议的工作将为研究锥虫基因表达的重要新工具提供重要的新工具，从而对转化控制在T. brucei中的作用以及对IT机制的线索以及有关单个基因产物的翻译程度（例如潜在药物靶标）的作用进行了全面看法。此外，它应该解决有关仅包含简短开放式读取框架的众多最近确定的RNA功能的当前辩论，并有可能识别非规范蛋白质编码的开放式读取框架，从而显着增强了持续的基因组注释。 项目叙述：寄生虫锥虫肿块会导致致命的人类非洲锥虫病（睡眠疾病）和药物治疗这种疾病是有毒的，并且面临抗药性。产生新药需要了解哪种蛋白质在寄生虫发育的引起疾病的阶段表达。与非感染阶段相比，该项目将应用一项新技术来测量感染性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标的新信息。