Guide RNA Binding Complex

描述(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。布鲁氏锥虫是非洲昏睡病的病原体，也是多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、发育重新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。由于目前使用的针对布氏毛滴虫的治疗方法无效且不安全，因此针对这种生物中唯一发现的细胞通路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，称为动泡体，它由几个上环和数千个微环组成。线粒体基因编码在Maxircle中，但大多数都是加密的：需要广泛的转录后尿苷插入/删除RNA编辑才能产生开放阅读框架。编辑反应的级联反应由嵌入~15亚单位RNA编辑核心复合体RECC(20S编辑小体)的酶催化，每一步都由小环编码的引导RNA(GRNAs)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、作用机制以及编辑后的代谢命运知之甚少。我们发现，成熟的gRNA是通过与gRNA结合复合体GRBC结合来稳定的，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，其功能超出了gRNA结合的范围。这项建议集中在GRBC的蛋白质组成和结构，gRNA-mRNA相互作用的机制，以及编辑后的gRNA置换。我们假设依赖于RNA底物的recc-GRBC组装代表了RNA编辑的全酶，并建议：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)确定对gRNA稳定和mRNA结合至关重要的GRBC亚基；以及4)剖析gRNA置换的机制。

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.