Functional definition of guide RNAs

指导RNA的功能定义

基本信息

批准号：
8968819
负责人：
Ruslan Afasizhev
金额：
$ 24.56万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-12-01 至 2016-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8968819
关键词：
Address Affinity African Trypanosomiasis American Antisense RNA Area Binding Biogenesis Biological Assay Biological Sciences Biology Cells Code Complex Computational algorithm Computer Simulation Data Developing Countries Elements Enzymes Eukaryota Event Evolution Exonuclease Foundations Future Gene Expression Generations Genetic Genetic Transcription Genome Genomics Global Change Guide RNA Health Kinetoplast DNA Knowledge Link Location Maps Messenger RNA Mitochondria Mitochondrial DNA Mitochondrial RNA Monitor Organism Outcome Parasites Parasitic Diseases Pathway interactions Pattern Play Point Mutation Population Process Proteins RNA RNA Binding RNA Editing RNA Precursors RNA Processing Resources Role Small RNA Stress System Time Transcript Transcription Initiation Site Transcriptional Regulation Trypanocidal Agents Trypanosoma Trypanosoma brucei brucei Uridine base genetic information genomic tools hemoflagellate insertion/deletion mutation knock-down mitochondrial genome mitochondrial messenger RNA novel therapeutics polyadenylated messenger RNA reconstitution reference genome relational database research study single molecule tool transcriptome transcriptome sequencing transcriptomics tripolyphosphate

项目摘要

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.

描述（申请人提供）：非洲昏睡病的病原体布氏锥虫是一种具有重大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，引导线粒体 mRNA 的 U 型插入/删除编辑的向导 RNA 的发现是这一广阔的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（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。该项目将从代表性菌株中生成完整的参考基因组，识别和绘制短 RNA 转录本，并根据计算机预测和功能分析注释指导和非指导 RNA。我们假设反义转录产生的非指导RNA在指导RNA前体的溶核过程中发挥重要作用，并建议：1）建立T. brucei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（SMRT）和双端合成测序（SBS）平台来组装和注释一组非冗余的小环，并重建大环的不同重复区域。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 末端加工和尿苷化，并将这些 RNA 映射到基因组位置。聚腺苷酸化 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。将开发计算机算法来根据已知和新识别的编辑模式来预测 gRNA。 2) 探索小RNA加工和稳定以及mRNA编辑之间的关系。我们将扰乱线粒体 RNA 加工中的特定步骤，并监测小 RNA 群体的变化，以根据指导 RNA 和非指导 RNA 参与编辑过程来区分它们。这些数据将与与编辑复合物结合的 RNA 和计算预测的 gRNA 进行交叉引用。