Functional definition of guide RNAs

描述（申请人提供）：非洲昏睡病的病原体布鲁氏锥虫是一种具有重要生物医学意义的单细胞寄生虫，也是研究小rna的重要实验系统。事实上，指导线粒体mRNA u插入/删除编辑的引导rna的发现是这一巨大生物学领域的第一个灯塔。然而，两个相互关联的领域仍然知之甚少：寄生虫线粒体基因组的复杂性，它由数千个小环和少数大环组成，以及线粒体DNA编码的小rna的功能。该项目将从一个具有代表性的菌株中生成一个完整的参考基因组，识别和绘制短RNA转录本，并根据计算机预测和功能分析对引导RNA和非引导RNA进行注释。我们假设由反义转录产生的非引导RNA在引导RNA前体的核分解加工中发挥了重要作用，并提出：1)建立布鲁氏体线粒体基因组和转录组的综合关系数据库。我们将使用单分子实时测序（SMRT）和合成对端测序（SBS）平台组装和注释一组非冗余的小环，并重建最大环的发散重复区。链特异性RNA-Seq方法将用于确定线粒体小rna的完整库，研究它们的32端加工和尿苷化，并将这些rna定位到基因组位置。聚腺苷化mrna的测序将用于评估RNA编辑过程的保真度，并确定最突出的编辑中间体、错误编辑和替代编辑事件。计算机算法将根据已知和新发现的编辑模式来预测grna。2)探索小RNA加工和稳定与mRNA编辑之间的关系。我们将干扰线粒体RNA加工的特定步骤，并监测小RNA群体的变化，根据它们参与编辑过程来区分引导RNA和非引导RNA。这些数据将与与编辑复合体结合的rna和计算预测的grna交叉引用。

DESCRIPTION (provided by applicant): The causative agent of African sleeping sickness, Trypanosoma brucei, is a unicellular parasite of major biomedical significance and is also an important experimental system for studying small RNAs. Indeed, the discovery of guide RNAs that direct U-insertion/deletion editing of mitochondrial mRNA was the first beacon in this immense area of biology. However, two interconnected areas remain poorly understood: the complexity of the parasite's mitochondrial genome, which is composed of thousands of minicircles and few maxicircles, and the functionality of small RNAs encoded by mitochondrial DNA. This project will generate a complete reference genome from a representative strain, identify and map short RNA transcripts and annotate guide and non- guide RNAs based on in silico predictions and functional assays. We hypothesize that non-guide RNAs produced by antisense transcription play an essential role in the nucleolytic processing of guide RNA precursors and propose to: 1) Build a comprehensive relational database of the mitochondrial genome and transcriptome in T. brucei. We will use the Single Molecule Real Time (SMRT) and paired-end sequencing by Synthesis (SBS) platforms to assemble and annotate a non-redundant set of minicircles and reconstitute the divergent repeat-containing region of the maxi circle. Strand-specific RNA-Seq approaches will be applied to determine a complete repertoire of small mitochondrial RNAs, investigate their 32 end processing and uridylation, and to map these RNAs to genomic locations. Sequencing of polyadenylated mRNAs will be used to assess the fidelity of RNA editing process and to establish the most prominent editing intermediates, misediting and alternative editing events. Computer algorithms will be developed to predict gRNAs based on known and newly-identified editing patterns. 2) Explore relationships between small RNA processing and stabilization, and mRNA editing. We will perturb specific steps in mitochondrial RNA processing and monitor changes in small RNA population to distinguish guide and non-guide RNAs based on their participation in the editing process. These data will be cross-referenced with RNAs bound to editing complexes and computationally-predicted gRNAs.