Guide RNA Binding Complex

描述（由申请人提供）：锥虫是在发展中国家造成重大健康危害的单细胞寄生虫。非洲昏睡病的病原体布鲁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，在这种寄生虫的巨大线粒体中已经发现了一些独特的基因表达途径，比如RNA编辑。由于目前采用的针对布鲁氏杆菌的治疗方法无效且不安全，因此靶向仅在该生物体中发现的细胞途径是一种有希望的治疗方法。锥虫线粒体包裹着一种不寻常的DNA结构，称为着丝体，它由几个最大环和数千个小环组成。线粒体基因以最大环编码，但大多数是加密的：需要广泛的转录后尿苷插入/删除RNA编辑来产生开放的阅读框。这些级联编辑反应是由嵌入到约15个亚基RNA编辑核心复合体RECC （20S编辑体）中的酶催化的，而每一步都是由微环编码的向导RNA （gRNAs）指导的。RECC的结构-功能研究取得了令人瞩目的进展，但对gRNA的生物发生、稳定性、与mRNA的结合、作用机制以及编辑后的代谢命运知之甚少。我们发现成熟的gRNA通过与gRNA结合复合体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，其功能可能超出gRNA的结合范围。本文将重点研究GRBC蛋白的组成和结构、gRNA- mrna相互作用的机制以及编辑后gRNA的位移。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中蛋白质-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位移的机制。

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.