Function of mRNA Editing in Trypanosomes

锥虫中 mRNA 编辑的功能

• 批准号: 7880344
• 负责人: STEPHEN L HAJDUK
• 金额: $ 3.45万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7880344
• 关键词: ATP synthase subunit 6; Africa South of the Sahara; African Trypanosomiasis; Antibodies; Biochemical Genetics; Biogenesis; Biology; Blood Circulation; Cell Fractionation; Characteristics; Cloning; Code; Complementary DNA; Complex; Computer Simulation; Cytochrome Oxidase Subunit III; Cytochrome c Reductase; DNA Binding; DNA Maintenance; Data; Databases; Development; Dominant-Negative Mutation; Electron Transport; Genes; Genetic; Goals; Guide RNA; In Vitro; Kinetoplast DNA; Lead; Mass Spectrum Analysis; Messenger RNA; Mitochondria; Mitochondrial Membrane Protein; Mitochondrial Proteins; Molecular; Mutagenesis; Nucleosides; Open Reading Frames; Organism; Oxidases; Pharmaceutical Preparations; Play; Process; Production; Proteins; Proteomics; RNA Editing; RNA Stability; Regulation; Respiratory System; Risk; Role; Sequence Analysis; Simulate; Site; Toxic effect; Transcript; Translating; Translations; Trypanosoma; Trypanosoma brucei brucei; Vaccines; Validation; abstracting; base; cDNA Library Construction; cytochrome c oxidase; drug development; genetic analysis; human disease; insertion/deletion mutation; mitochondrial membrane; novel; polypeptide; positional cloning; protein complex; protein function; respiratory; simulation

Abstract RNA editing in trypanosomes is a remarkable post-transcriptional process that results in the formation of mitochondrial mRNAs differing from their genes by the insertion or deletion of uridylyl (U) nucleosides. The information for RNA editing is provided by small guide RNAs (gRNAs) that basepair with their cognate pre-mRNAs to direct the precise sites for U-insertion or U-deletion. While considerable progress has been made in the elucidation of the components of the editing machinery and the general mechanism of RNA editing little is known about the regulation and function of this process. Simplistically, it has been thought that the sole function for RNA editing was to correct mistakes in mitochondrial protein coding genes thus allowing the translation of edited mRNAs to produce components of the mitochondrial respiratory system. While this is certainly an important function for RNA editing we recently discovered that primary mRNAs are differentially edited and translated to produce novel mitochondrial proteins. Our initial studies focused on the mRNA for cytochrome c oxidase III (COIII), which we discovered was alternatively edited in Trypanosoma brucei. A gRNA for the alternatively edited COIII transcript was identified and antibodies against the predicted novel coding sequence of the alternatively edited protein (AEP-1) reacted with a mitochondrial membrane protein. We have recently expanded the analysis of alternative mRNA editing to four additional genes, NADH dehydrogenase subunits 7, 8, 9 (ND7, 8, 9) and ATP synthase subunit 6 (A6). Alternatively edited mRNAs, creating novel open reading frames, were found for each of these genes. The overall goals of this proposal are to determine the extent of alternative mRNA editing in trypanosomes and the function mitochondrial proteins encoded by these RNAs. To accomplish these goals the following specific aims are proposed. In Specific Aim 1, we will examine the function of AEP-1 using a combination of biochemical and genetic approaches. In these studies, we will use dominant- negative mutants to evaluate the role of AEP-1 in kinetoplast DNA maintenance, proteomic analysis to identify associated proteins and in vitro DNA binding studies to further explore the function of this protein. Specific Aim 2, we will carryout a comprehensive gRNA and mRNA sequence analysis to examine the extent of potential mitochondrial protein diversity generated by alternative mRNA editing. Using conventional cDNA cloning and sequencing of mitochondrial mRNAs and 454 sequencing of gRNAs we will evaluate the extent of alternative RNA editing for mRNAs encoded by 12 mitochondrial genes. These data will be used to generate in silico RNA editing simulations to predict the magnitude of alternative mRNA editing. Putative alternatively edited mRNAs will be validated based on coding sequence, RNA stability and the identification of predicted protein products by proteomic, cell fractionation and reverse genetics. Together these studies will provide the first analysis of the role of RNA editing in protein diversification in trypanosomes and is likely to lead to the discovery of novel mitochondrial proteins.

抽象的 锥虫中的RNA编辑是一个非凡的转录后过程，导致形成 线粒体mRNA与其基因不同，尿液插入或缺失（U） 核苷。 RNA编辑的信息由小导向RNA（GRNA）提供 用它们的同源前MRNA指导u插入或u-Deption的精确位点。尽管 在阐明编辑机器的组件中取得了长足的进步 RNA编辑的一般机制对此的调节和功能知之甚少 过程。简单地说，人们认为RNA编辑的唯一函数是纠正 线粒体蛋白编码基因的错误，从而使编辑的mRNA翻译成 产生线粒体呼吸系统的组成部分。虽然这当然很重要 RNA编辑的功能我们最近发现原发性mRNA是差异编辑的，并且 翻译成产生新的线粒体蛋白。我们的最初研究集中于mRNA 我们发现的细胞色素C氧化酶III（COIII）是在Brucei锥虫瘤中编辑的。 确定了用于替代编辑的COIII转录本的GRNA，并针对预测的抗体 替代编辑蛋白（AEP-1）的新颖编码顺序与线粒体反应 膜蛋白。我们最近将替代mRNA编辑的分析扩展到四个 其他基因，NADH脱氢酶亚基7、8、9（ND7、8、9）和ATP合酶亚基6（A6）。 另外，发现了这些基因中的每个基因的编辑mRNA，创建了新颖的开放式阅读框。 该提案的总体目标是确定在 这些RNA编码的锥虫和线粒体蛋白的功能。完成这些 目标提出了以下特定目标。在特定目标1中，我们将检查AEP-1的功能 结合生化方法和遗传方法。在这些研究中，我们将使用主要 负突变体评估AEP-1在动力学DNA维持中的作用，蛋白质组学分析至 鉴定相关的蛋白质和体外DNA结合研究，以进一步探索其功能 蛋白质。具体的目标2，我们将将全面的GRNA和mRNA序列分析带到 检查替代mRNA编辑产生的潜在线粒体蛋白多样性的程度。 使用线粒体mRNA和454测序的常规cDNA克隆和测序 GRNA我们将评估由12个线粒体编码的mRNA的替代RNA编辑程度 基因。这些数据将用于在硅RNA编辑模拟中生成，以预测 替代mRNA编辑。推定对编码的编辑的编辑mRNA将得到验证 蛋白质组学细胞序列，RNA稳定性和鉴定预测的蛋白质产物 分馏和反向遗传学。这些研究将共同​​提供首先分析的作用 RNA在锥虫中蛋白质多样化中的编辑，很可能导致发现新颖 线粒体蛋白。