Simpson Rachel M, Bruno Andrew E, Bard Jonathan E, Buck Michael J, Read Laurie K
Department of Microbiology and Immunology, University at Buffalo, Jacobs School of Medicine and Biomedical Sciences, Buffalo, New York 14214, USA.
Center for Computational Research, University at Buffalo, Buffalo, New York 14203, USA.
RNA. 2016 May;22(5):677-95. doi: 10.1261/rna.055160.115. Epub 2016 Feb 23.
Uridine insertion/deletion RNA editing in kinetoplastids entails the addition and deletion of uridine residues throughout the length of mitochondrial transcripts to generate translatable mRNAs. This complex process requires the coordinated use of several multiprotein complexes as well as the sequential use of noncoding template RNAs called guide RNAs. The majority of steady-state mitochondrial mRNAs are partially edited and often contain regions of mis-editing, termed junctions, whose role is unclear. Here, we report a novel method for sequencing entire populations of pre-edited partially edited, and fully edited RNAs and analyzing editing characteristics across populations using a new bioinformatics tool, the Trypanosome RNA Editing Alignment Tool (TREAT). Using TREAT, we examined populations of two transcripts, RPS12 and ND7-5', in wild-typeTrypanosoma brucei We provide evidence that the majority of partially edited sequences contain junctions, that intrinsic pause sites arise during the progression of editing, and that the mechanisms that mediate pausing in the generation of canonical fully edited sequences are distinct from those that mediate the ends of junction regions. Furthermore, we identify alternatively edited sequences that constitute plausible alternative open reading frames and identify substantial variability in the 5' UTRs of both canonical and alternatively edited sequences. This work is the first to use high-throughput sequencing to examine full-length sequences of whole populations of partially edited transcripts. Our method is highly applicable to current questions in the RNA editing field, including defining mechanisms of action for editing factors and identifying potential alternatively edited sequences.
动质体中的尿苷插入/缺失RNA编辑需要在线粒体转录本的全长范围内添加和删除尿苷残基，以生成可翻译的mRNA。这个复杂的过程需要协同使用几种多蛋白复合物，以及顺序使用称为引导RNA的非编码模板RNA。大多数稳态线粒体mRNA是部分编辑的，并且通常包含编辑错误的区域，称为连接点，其作用尚不清楚。在这里，我们报告了一种新方法，用于对预编辑、部分编辑和完全编辑的RNA的整个群体进行测序，并使用一种新的生物信息学工具——锥虫RNA编辑比对工具（TREAT）分析群体间的编辑特征。使用TREAT，我们检测了野生型布氏锥虫中两个转录本RPS12和ND7-5'的群体。我们提供的证据表明，大多数部分编辑的序列包含连接点，在编辑过程中会出现内在的暂停位点，并且在生成标准完全编辑序列时介导暂停的机制与介导连接点区域末端的机制不同。此外，我们鉴定了构成合理的替代开放阅读框（ORF）的可变编辑序列，并鉴定了标准和可变编辑序列5'非翻译区（UTR）中的大量变异性。这项工作是首次使用高通量测序来检测部分编辑转录本整个群体的全长序列。我们的方法高度适用于RNA编辑领域当前的问题，包括确定编辑因子的作用机制和识别潜在的可变编辑序列。
