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.

摘要 锥虫中的RNA编辑是一个显著的转录后过程， 通过插入或缺失尿苷酰（U）而与其基因不同的线粒体mRNA 核苷RNA编辑的信息由碱基配对的小向导RNA（gRNA）提供。 与它们的同源前体mRNA一起指导U插入或U缺失的精确位点。而 在阐明编辑机制的组成部分方面已经取得了相当大的进展 而RNA编辑的一般机制对其调控和功能知之甚少， 过程简单地说，人们认为RNA编辑的唯一功能是纠正 线粒体蛋白质编码基因中的错误，从而允许编辑的mRNA翻译， 产生线粒体呼吸系统的组成部分。虽然这无疑是一个重要的 我们最近发现，主要的mRNA是差异编辑的， 翻译产生新的线粒体蛋白。我们最初的研究集中在mRNA上， 细胞色素c氧化酶III（COIII），我们发现其在布氏锥虫中被交替编辑。 鉴定了替代性编辑的COIII转录物的gRNA，并鉴定了针对预测的COIII转录物的抗体。 与线粒体反应的交替编辑蛋白（AEP-1）的新编码序列 膜蛋白我们最近将替代mRNA编辑的分析扩展到四个 另外的基因，NADH脱氢酶亚基7、8、9（ND 7、8、9）和ATP合酶亚基6（A6）。 对于这些基因中的每一个，都发现了可选择地编辑的mRNA，产生了新的开放阅读框架。 这项提案的总体目标是确定在基因组中替代mRNA编辑的程度。 锥虫和这些RNA编码的功能线粒体蛋白。完成这些 目标提出了以下具体目标。在具体目标1中，我们将研究AEP-1的功能 使用生物化学和遗传学方法的组合。在这些研究中，我们将使用显性- 阴性突变体，以评估AEP-1在动质体DNA维持中的作用，蛋白质组学分析， 鉴定相关蛋白和体外DNA结合研究，以进一步探索这种功能， 蛋白具体目标2，我们将进行全面的gRNA和mRNA序列分析， 检查由替代mRNA编辑产生的潜在线粒体蛋白质多样性的程度。 使用常规的cDNA克隆和线粒体mRNA的测序以及454个测序， 我们将评估由12种线粒体基因编码的mRNA的替代RNA编辑的程度。 基因.这些数据将用于生成计算机RNA编辑模拟，以预测 替代mRNA编辑。将基于编码验证推定的替代编辑的mRNA。 序列、RNA稳定性和通过蛋白质组学、细胞 分馏和反向遗传学。这些研究将共同提供第一个分析的作用， RNA编辑在锥虫蛋白质多样化中的作用，并可能导致发现新的 线粒体蛋白