Guide RNA Binding Complex

描述（由申请人提供）：锥虫是单细胞寄生虫，在发展中国家造成重大健康危害。 非洲昏睡病的病原体布氏锥虫也是几个研究领域的重要模式生物，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。事实上，一些独特的基因表达途径，如RNA编辑，已经在这种寄生虫的巨大寄生虫中发现。由于目前针对T.布鲁氏菌是无效和不安全的，靶向仅在这种生物体中发现的细胞途径是一种有前途的治疗方法。锥虫幼虫体内有一种不寻常的DNA结构，称为动基体，由几个大环和数千个小环组成。线粒体基因在大环中编码，但大多数是加密的：需要广泛的转录后尿苷插入/缺失RNA编辑来产生开放阅读框。编辑反应的级联由嵌入到~15亚基RNA编辑核心复合物（20 S编辑体）中的酶催化，而每个步骤由小环编码的向导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、作用机制和编辑后的代谢命运知之甚少。我们已经发现成熟gRNA通过与gRNA结合复合物GRBC结合而稳定，并且已经鉴定了直接负责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，其功能超出了gRNA结合。该提案的重点是GRBC蛋白的组成和结构，gRNA-mRNA相互作用的机制，以及编辑后的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）描述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gRNA稳定性和mRNA结合所必需的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是有毒和无效的，迫切需要新的药物。 由于线粒体基因表达对寄生虫至关重要，靶向必需的寄生虫特异性核糖核蛋白复合物，如指导RNA结合复合物（GRBC），是开发新的杀锥虫剂的潜在方法。

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.