Functional definition of guide RNAs

描述（由申请人提供）：非洲昏睡病的病原体布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的U插入/缺失编辑的指导RNA的发现是生物学这一巨大领域的第一个灯塔。然而，两个相互关联的领域仍然知之甚少：寄生虫的线粒体基因组的复杂性，它是由数千个小环和几个大环组成，以及线粒体DNA编码的小RNA的功能。该项目将从代表性菌株中生成完整的参考基因组，鉴定和定位短RNA转录物，并基于计算机预测和功能测定注释指导和非指导RNA。我们假设反义转录产生的非向导RNA在向导RNA前体的溶核加工中起重要作用，并提出：1）建立一个全面的T.布鲁塞。我们将使用单分子真实的时间（SMRT）和合成配对末端测序（SBS）平台来组装和注释一组非冗余的微环，并重建大环的含有发散重复序列的区域。链特异性RNA-Seq方法将用于确定小线粒体RNA的完整库，研究其32端加工和尿苷化，并将这些RNA映射到基因组位置。多聚腺苷酸化mRNA的测序将用于评估RNA编辑过程的保真度，并建立最突出的编辑中间体、错配和替代编辑事件。将开发计算机算法来基于已知和新鉴定的编辑模式预测gRNA。2)探索小RNA加工和稳定化与mRNA编辑之间的关系。我们将干扰线粒体RNA加工中的特定步骤，并监测小RNA群体的变化，以根据它们参与编辑过程来区分指导和非指导RNA。这些数据将与结合到编辑复合物的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.