Guide RNA Binding Complex

引导RNA结合复合物

基本信息

批准号：
8342887
负责人：
Ruslan Afasizhev
金额：
$ 18.29万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
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 2013-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8342887
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 Delivery Systems 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. PUBLIC HEALTH RELEVANCE: Trypanosomes and closely related organisms are the causative agents of parasitic diseases in developing countries. Available treatments are often toxic and ineffective, and the need for new drugs is pressing. Because mitochondrial gene expression is vital for the parasite, targeting essential parasite-specific ribonucleoprotein complexes, such as the guide RNA binding complex (GRBC), is a potential approach toward developing new trypanocides.

描述（由申请人提供）：锥虫是单细胞寄生虫，对发展中国家的健康造成重大危害。非洲昏睡病的病原体布氏锥虫也是抗原变异、宿主与病原体相互作用、发育重编程和线粒体生物学等多个研究领域的重要模式生物。事实上，在这种寄生虫的巨型线粒体中已经发现了一些独特的基因表达途径，例如RNA编辑。由于目前针对布氏锥虫的治疗方法无效且不安全，因此针对这种生物体中独有的细胞途径是一种有前途的治疗方法。锥虫线粒体包围着一种不寻常的 DNA 结构，称为动质体，它由几个大环和数千个小环组成。线粒体基因在大环中编码，但大多数都是加密的：需要进行广泛的转录后尿苷插入/删除 RNA 编辑才能产生开放阅读框。编辑反应的级联由嵌入约 15 个亚基 RNA 编辑核心复合体 RECC（20S 编辑体）中的酶催化，而每个步骤均由小环编码的引导 RNA (gRNA) 指导。 RECC 的结构功能研究取得了令人印象深刻的进展，但对 gRNA 的生物发生、稳定性、与 mRNA 的结合、作用机制和编辑后代谢命运知之甚少。我们发现成熟的 gRNA 通过与 gRNA 结合复合物 GRBC 结合而稳定，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 RECC，并且其功能超出了 gRNA 结合范围。该提案重点关注 GRBC 蛋白组成和结构、gRNA-mRNA 相互作用机制以及编辑后 gRNA 置换。我们假设RNA底物依赖性RECC-GRBC组装代表RNA编辑全酶，并提出：1）描绘GRBC内蛋白质-蛋白质和RNA介导的相互作用； 2）阐明GRBC-RECC相互作用的功能作用； 3) 鉴定对于 gRNA 稳定性和 mRNA 结合至关重要的 GRBC 亚基； 4) 剖析 gRNA 置换的机制。公共卫生相关性：锥虫和密切相关的生物体是发展中国家寄生虫病的病原体。现有的治疗方法往往有毒且无效，因此迫切需要新药。由于线粒体基因表达对于寄生虫至关重要，因此靶向寄生虫特异性核糖核蛋白复合物（例如指导 RNA 结合复合物 (GRBC)）是开发新锥虫杀剂的潜在方法。