Deep sequence profiling of gRNA transcriptomes in two stages of Trypanosoma bruce

布鲁斯锥虫两个阶段 gRNA 转录组的深度序列分析

• 批准号: 8233286
• 负责人: DONNA Jay KOSLOWSKY
• 金额: $ 7.6万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 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 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8233286
• 关键词: Affect; African Trypanosomiasis; Attention; Binding Sites; Blood Circulation; Chagas Disease; Communities; Complement; Complex; Data; Development; Dissection; Drug Delivery Systems; Elements; Employee Strikes; Energy Metabolism; Environment; Evolution; Free Energy; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Proteins; Generations; Genes; Genetic Transcription; Genome; Guide RNA; In Vitro; Initiator Codon; Insecta; Intervention; Knowledge; Lead; Leishmaniasis; Libraries; Life Cycle Stages; Messenger RNA; Methods; Mitochondria; Modeling; Molecular Profiling; Open Reading Frames; Parasites; Parasitic Diseases; Pattern; Play; Population; Process; Proteins; Protozoa; RNA; RNA Databases; RNA Editing; RNA chemical synthesis; RNA library; Reading; Regulation; Regulator Genes; Research; Role; Site; Small RNA; Staging; Structure; System; Tail; Techniques; Technology; Terminator Codon; Transcript; Transcription Process; Trypanosoma; Trypanosoma brucei brucei; World Health Organization; base; density; insertion/deletion mutation; insight; mathematical model; mitochondrial genome; mitochondrial messenger RNA; public health relevance; uridylate

DESCRIPTION (provided by applicant): One of the most striking and ancient examples of small RNA regulation of gene expression is the process of RNA editing in the mitochondria of trypanosomes. In these parasites, RNA editing involves extensive uridylate insertions and deletions within most of the mitochondrial mRNAs. Over 1200 small guide RNAs (gRNAs) are predicted to be responsible for directing the sequence changes that create start and stop codons, correct frameshifts and for many of the mRNAs, generate most of the open reading frame of the mRNA. In addition, alternative editing appears to be extensive and creates the opportunity for unprecedented protein diversity. Guide RNAs are key components of the RNA editing process; information on the total number and transcript levels of the gRNAs involved in this complex process would provide key information necessary for our understanding of its regulation. Despite many conventional sequencing attempts, large numbers of the gRNAs needed for the extensive editing of the protein genes are still unidentified. In addition, no information on gRNA expression levels exists. The recent development of second generation, ultra-high-throughput sequencing platforms allow both the identification of rare transcripts and the quantification of specific RNA levels based on the density of corresponding reads. The objective of this proposal is to utilize deep sequencing technology to characterize the full gRNA transcriptomes in the two main life cycle stages of Trypanosoma brucei. High-throughput sequencing will allow the identification of all gRNAs within a population, provide quantitative information on their expression levels and determine the transcriptional structure of the gRNA genes. Characterization of the full complement of gRNAs needed for both conventional and alternative editing, will allow the identification of the multiple target sites within each mRNA. With this information we can begin to predict the free energy requirements for the multiple gRNA/mRNA interactions necessary for the editing of a single transcript. These data will provide insight into both the developmental regulation of editing and the selective use of alternative guide RNAs. In addition, knowledge of the transcriptional structure of gRNAs may allow the identification of specific minicircle sequence elements important for transcription and processing. Small RNAs are now known to be key regulators of gene expression. The ability of small RNAs to accurately and efficiently basepair with their specific targets is crucial to their function. Dissection of the targeting strategies used in this ancient RNA editing system, may provide insight into the evolution of small RNA targeting strategies. PUBLIC HEALTH RELEVANCE: The kinetoplastid protozoa include the causative agents for three of the most severe parasitic diseases targeted for attention by the World Health Organization (WHO); Leishmaniasis, sleeping sickness and Chagas disease. Together these parasites affect approximately 550 million people in the developing world. RNA editing is essential for mitochondrial function and is unique to these parasites making this process a good target for drug intervention.

描述（由申请人提供）：小RNA调控基因表达的最引人注目和最古老的例子之一是锥虫线粒体中的RNA编辑过程。在这些寄生虫中，RNA编辑涉及大多数线粒体mRNA内的广泛尿苷酸插入和缺失。预计超过1200种小向导RNA（gRNA）负责指导产生起始和终止密码子、校正移码的序列变化，并且对于许多mRNA，产生mRNA的大部分开放阅读框。此外，替代编辑似乎是广泛的，并创造了前所未有的蛋白质多样性的机会。向导RNA是RNA编辑过程的关键组成部分;关于参与这一复杂过程的gRNA的总数和转录水平的信息将为我们理解其调控提供必要的关键信息。尽管进行了许多常规测序尝试，但蛋白质基因的广泛编辑所需的大量gRNA仍然未被识别。此外，还不存在有关gRNA表达水平的信息。第二代超高通量测序平台的最新发展允许基于相应读数的密度鉴定罕见转录本和定量特定RNA水平。该提案的目的是利用深度测序技术来表征布氏锥虫两个主要生命周期阶段中的全gRNA转录组。高通量测序将允许鉴定群体内的所有gRNA，提供关于其表达水平的定量信息并确定gRNA基因的转录结构。常规和替代编辑所需的gRNA的完整互补的表征将允许鉴定每个mRNA内的多个靶位点。有了这些信息，我们可以开始预测编辑单个转录物所需的多种gRNA/mRNA相互作用的自由能需求。这些数据将提供深入了解编辑的发育调控和选择性使用替代向导RNA。此外，gRNA的转录结构的知识可以允许鉴定对转录和加工重要的特定小环序列元件。现在已知小RNA是基因表达的关键调节因子。小RNA与其特异性靶标准确有效地碱基配对的能力对其功能至关重要。对这个古老的RNA编辑系统中使用的靶向策略的剖析，可以提供对小RNA靶向策略进化的洞察。 公共卫生关系：动体原虫包括世界卫生组织（世卫组织）关注的三种最严重寄生虫病的病原体：利什曼病、昏睡病和恰加斯病。这些寄生虫共影响发展中国家约5.5亿人。RNA编辑对于线粒体功能至关重要，并且是这些寄生虫所独有的，使得这一过程成为药物干预的良好靶点。