Function of mRNA Editing in Trypanosomes

摘要 锥虫体内的RNA编辑是一个显著的转录后过程，导致了 由于插入或缺失尿酰(U)而与其基因不同的线粒体mRNAs 核苷。用于RNA编辑的信息由基本的小引导RNA(GRNAs)提供 与它们的同源前mRNAs一起指导U-插入或U-缺失的精确位点。而当 在阐明编辑机械的组成部分方面取得了相当大的进展 而RNA编辑的一般机制对此的调节和功能知之甚少 进程。简单地说，人们一直认为RNA编辑的唯一功能是纠正 线粒体蛋白编码基因的错误，从而允许编辑的mRNA翻译成 产生线粒体呼吸系统的组成部分。虽然这肯定是一个重要的 用于RNA编辑的功能我们最近发现，初级mRNA是经过差异编辑的，并且 翻译成新的线粒体蛋白。我们最初的研究集中在信使核糖核酸上 细胞色素c氧化酶III(COIII)，我们发现它在布鲁氏锥虫中是交替编辑的。 鉴定了交替编辑的COIII转录本的gRNA，并针对预测的 交替编辑蛋白(AEP-1)的新编码序列与线粒体反应 膜蛋白。我们最近将可供选择的信使核糖核酸编辑的分析扩大到四个 其他基因，NADH脱氢酶亚基7、8、9(ND7、8、9)和ATP合成酶亚基6(A6)。 对于这些基因中的每一个，都发现了编辑过的mRNAs，它们创造了新的开放阅读框架。 这项建议的总体目标是确定可供选择的mRNA编辑的程度 锥虫和由这些RNA编码的功能线粒体蛋白。要实现这些目标 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 使用生化和遗传方法的组合。在这些研究中，我们将使用优势- 阴性突变体评估AEP-1在动质粒DNA维持中的作用，蛋白质组学分析 确定相关蛋白和体外DNA结合研究，以进一步探索这一功能 蛋白。具体目标2，我们将对gRNA和mRNA序列进行全面的分析 检查由替代的信使核糖核酸编辑产生的潜在线粒体蛋白质多样性的程度。 使用常规的线粒体mRNAs的cDNA克隆和测序以及454测序 我们将评估对由12个线粒体编码的mRNAs进行替代RNA编辑的程度 基因。这些数据将用于在Silico RNA编辑模拟中生成以预测 可选的信使核糖核酸编辑。将根据编码验证假定的或编辑的mRNAs 序列、RNA稳定性和蛋白质组、细胞预测蛋白产物的鉴定 分离和反向遗传学。总之，这些研究将提供第一次分析 在锥虫蛋白质多样化中的RNA编辑，并可能导致新的发现 线粒体蛋白质。

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.