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（由申请人提供）：非洲睡眠疾病的病因Brucei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。实际上，发现指导RNA直接直接的U插入/缺失编辑是线粒体mRNA是这一巨大生物学领域的第一个信标。然而，两个相互联系的区域仍然鲜为人知：寄生虫的线粒体基因组的复杂性，该基因组由数千个微圆和少数最大圆形组成，以及由线粒体DNA编码的小RNA的功能。该项目将从代表性菌株，识别和绘制简短的RNA转录本，注释指南和非指导RNA中产生一个完整的参考基因组，并基于计算机预测和功能测定中的非指导RNA。我们假设反义转录产生的非诱导RNA在指南RNA前体的核解过程中起着至关重要的作用，并提出：1）在T. Brucei中构建线粒体基因组和转录组的全面关系数据库。我们将使用合成平台（SBS）平台使用单分子实时（SMRT）和配对结束来组装和注释一组非冗余的小圆圈，并重构Maxi Circle的不同重复区域。将应用链特异性RNA-seq方法来确定小线粒体RNA的完整曲目，研究其32端处理和尿苷，并将这些RNA映射到基因组位置。多腺苷酸化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，误用和替代编辑事件。将开发计算机算法以根据已知和新识别的编辑模式预测GRNA。 2）探索小的RNA处理与稳定和mRNA编辑之间的关系。我们将在小rNA群体中的线粒体RNA处理和监测变化中的特定步骤，以根据其参与编辑过程来区分指南和非指南RNA。这些数据将与与编辑复合物和计算预测的GRNA结合的RNA进行交叉引用。