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

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编辑涉及在大多数线粒体mrna中广泛的尿苷酸插入和删除。据预测，超过1200个小向导rna （gRNAs）负责指导序列变化，产生起始和停止密码子，纠正帧移位，对许多mRNA来说，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，但大量用于蛋白质基因广泛编辑的grna仍未被识别。此外，没有关于gRNA表达水平的信息。最近发展的第二代超高通量测序平台既可以鉴定稀有转录本，也可以根据相应reads的密度定量特定RNA水平。本提案的目的是利用深度测序技术来表征布鲁氏锥虫两个主要生命周期阶段的完整gRNA转录组。高通量测序将允许鉴定群体内的所有gRNA，提供其表达水平的定量信息，并确定gRNA基因的转录结构。对传统和替代编辑所需的grna的完整补体进行表征，将允许识别每个mRNA中的多个靶点。有了这些信息，我们就可以开始预测编辑单个转录本所需的多个gRNA/mRNA相互作用的自由能需求。这些数据将为编辑的发育调控和替代向导rna的选择性使用提供见解。此外，了解grna的转录结构可能有助于鉴定对转录和加工重要的特定微环序列元件。现在已知小rna是基因表达的关键调节因子。小rna准确有效地与特定靶标进行碱基对的能力对其功能至关重要。解剖这种古老的RNA编辑系统中使用的靶向策略，可能为小RNA靶向策略的进化提供见解。

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.