Guide RNA Binding Complex

引导RNA结合复合物

基本信息

批准号：
8466926
负责人：
Ruslan Afasizhev
金额：
$ 38.47万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-10 至 2017-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8466926
关键词：
Affinity Africa African Trypanosomiasis Animal Model Antigenic Variation Architecture Area Basic Science Binding Biochemical Biogenesis Biology Cell Line Charge Code Complex DNA Structure Data Developing Countries Development Diphosphates Drug Targeting Enzymes Gene Expression Genes Genetic Genome Guide RNA Health Health Hazards Holoenzymes Hybrids Hydrolase Hydrolysis In Vitro Lead Link Mediating Messenger RNA Metabolic Mitochondria Mitochondrial RNA Nuclear Open Reading Frames Organism Parasites Parasitic Diseases Pathway interactions Pharmaceutical Preparations Phosphorylation Polyadenylation Process Proteins Proteomics RNA RNA Binding RNA Editing RNA Helicase RNA Interference RNA Processing RNA Stability RNA annealing RNA-Binding Proteins Reaction Recruitment Activity Repression Research Ribonucleoproteins Role Structure Testing Therapeutic Transcript Translations Trypanocidal Agents Trypanosoma Trypanosoma brucei brucei Uridine cellular targeting helicase in vivo insertion/deletion mutation pathogen prevent protein complex reconstitution

项目摘要

DESCRIPTION (provided by applicant): Trypanosomes are unicellular parasites responsible for major health hazards in developing countries. The causative agent of African sleeping sickness, Trypanosoma brucei, is also an important model organism for several areas of research, including antigenic variation, host-pathogen interaction, developmental reprogramming, and mitochondrial biology. Indeed, some unique gene expression pathways, such as RNA editing, have been discovered in this parasite's giant mitochondrion. Because currently employed treatments against T. brucei are ineffective and unsafe, targeting cellular pathways that are found exclusively in this organism is a promising therapeutic approach. The trypanosome mitochondrion encloses an unusual DNA structure, called the kinetoplast, which is composed of few maxicircles and thousands of minicircles. Mitochondrial genes are encoded in maxicircles, but most are encrypted: an extensive post-transcriptional uridine insertion/deletion RNA editing is required to produce open reading frames. The cascade of editing reactions is catalyzed by enzymes embedded into the ~15-subunit RNA editing core complex, RECC (20S editosome), while each step is directed by minicircle-encoded guide RNAs (gRNAs). Structure-function studies of RECC achieved impressive progress, but little is known about gRNA biogenesis, stabilization, binding to mRNA, mechanism of action, and post-editing metabolic fate. We have discovered that mature gRNAs are stabilized via association with the gRNA binding complex, GRBC, and have identified the two subunits directly responsible for gRNA binding. Preliminary studies indicate that GRBC's complexity likely exceeds that of the RECC and that its functions extend beyond gRNA binding. This proposal focuses on GRBC protein composition and architecture, mechanisms of gRNA-mRNA interaction, and post-editing gRNA displacement. We hypothesize that RNA substrate-dependent RECC-GRBC assembly represents the RNA editing holoenzyme and propose to: 1) delineate protein-protein and RNA-mediated interactions within GRBC; 2) elucidate the functional role of GRBC-RECC interaction; 3) identify GRBC subunits essential for gRNA stability and mRNA binding; and 4) dissect the mechanism of gRNA displacement.

描述（由申请人提供）：锥虫是负责发展中国家重大健康危害的单细胞寄生虫。非洲睡眠疾病的病因Brucei锥虫也是多种研究领域的重要模型生物，包括抗原变异，宿主 - 病原体相互作用，发育重编程和线粒体生物学。实际上，在该寄生虫的巨型线粒体中发现了一些独特的基因表达途径，例如RNA编辑。由于目前针对布鲁氏菌的治疗方法无效且不安全，因此针对这种生物体中仅发现的细胞途径是一种有希望的治疗方法。锥虫线粒体包围了一种不寻常的DNA结构，称为动力体，该结构由少量的最大圆形和数千个微量圆形组成。线粒体基因在Maxicircles中编码，但大多数是加密的：需要进行广泛的转录后尿苷插入/缺失RNA编辑以产生开放的阅读框架。通过嵌入〜15个亚基RNA编辑核心复合物，RECC（20S编辑体）的酶催化了级联反应的级联反应，而每个步骤均由微米编码的指南RNA（GRNA）指导。 RECC的结构 - 功能研究取得了令人印象深刻的进步，但对GRNA生物发生，稳定，与mRNA结合，作用机理和编辑后代谢命运知之甚少。我们已经发现，成熟的GRNA通过与GRNA结合复合物GRBC的结合稳定，并确定了直接负责GRNA结合的两个亚基。初步研究表明，GRBC的复杂性可能超过了RECC的复杂性，其功能范围超出了GRNA结合。该建议的重点是GRBC蛋白组成和结构，GRNA-MRNA相互作用的机制以及编辑后的GRNA位移。我们假设RNA底物依赖性RECC-GRBC组件代表RNA编辑全酶，并提出：1）DELINEATE蛋白 - 蛋白质 - 蛋白质蛋白质和RNA介导的相互作用在GRBC中； 2）阐明GRBC-RECC相互作用的功能作用； 3）确定GRBC亚基对于GRNA稳定性和mRNA结合所必需的； 4）剖析GRNA位移的机理。