Function of the Kinetoplast in the Hemoflagellates

描述（由申请人提供）：u插入/删除RNA编辑是发生在锥虫寄生虫线粒体中的一种独特的转录后RNA修饰过程。编辑通过在特定位点插入和删除尿苷，修改从线粒体DNA“隐基因”转录的16个线粒体mrna中的12个序列，使其可翻译。我们发现了一类新的短RNA（引导RAs或gRNAs），它们包含通过碱基配对编辑的序列信息，使这种类型的RNA修饰符合中心法则。我们提出的基于grna介导的假设编辑模型涉及多个3‘-5’周期的切割、u -添加或u -删除和连接。我们为该模型提出的负责酶活性的蛋白质被鉴定为1个多蛋白复合物RNA编辑核心复合物或RECC的组成部分。另外一些可能参与RNA调控的多蛋白复合物通过RNA连接物与RECC相互作用。编辑发生在着丝质体谱系的所有成员中，包括几种人类疾病的致病因子锥虫寄生虫。如果不了解编辑复合物的分子结构及其与底物rna的相互作用，就无法理解编辑。我们建议对编辑反应的主要成分- 1md RECC进行高分辨率的结构-功能分析。我们将尝试通过在原子分辨率上确定这些颗粒的三维结构，并识别编辑反应中不同功能状态下的蛋白质成分及其蛋白质-蛋白质和蛋白质- rna相互作用，来确认和扩展我们假设的编辑反应模型。分子结构信息将从这一途径的详细机制方面进行解释，这些知识将为未来研究全酶和rna提供基础编辑与线粒体RNA调控和蛋白质翻译的可能相互作用。这一点很重要，因为除了RNA编辑之外，锥虫已经成为研究许多基本生物现象的重要真核模型系统，如蛋白质表达、反式剪接表面抗原变异和鞭毛运动。由于现在看来线粒体RNA编辑可能与翻译和RNA调控相互作用，因此了解编辑将有助于锥虫作为模式生物系统的发展。此外，u插入/删除编辑作为多种其他转录后RNA修饰现象的范例，如人脑中折叠引导的a到I编辑，sno-RNA引导的核糖体RNA甲基化和假尿嘧啶化，甚至RNAi中Si -RNA引导的mrna切割，本项目获得的信息可能与理解这些其他重要的生物学现象有关。最后但并非最不重要的是，编辑途径对于生存能力至关重要，并且可能为开发针对这些寄生虫引起的疾病的干预措施提供潜在目标。

DESCRIPTION (provided by applicant): U-insertion/deletion RNA editing is a unique post-transcriptional RNA modification process occurring in the mitochondria of trypanosomatid parasites. Editing modifies the sequences of 12 of 16 mitochondrial mRNAs transcribed from mitochondrial DNA "cryptogenes" by insertion and deletion of uridines at specific sites to render them translatable. Our discovery of a new class of short RNAs (guide RAs or gRNAs) that contain the sequence information for editing by base pairing in trans brought this type of RNA modification into line with the central dogma. The hypothetical editing model we proposed based on the mediation of gRNAs involved multiple 3'-5' cycles of cleavage, U-addition or U-deletion and ligation. The proteins responsible for the enzymatic activities we proposed for this model were identified as components of a 1 md multiprotein complex, the RNA Editing Core Complex or RECC. Several additional multiprotein complexes which may be involved in RNA regulation interact with the RECC via RNA linkers. Editing occurs in all members of the Kinetoplastid lineage, which includes the trypanosomatid parasites, the causal agents of several human diseases. The understanding of editing cannot proceed without knowledge of the molecular architecture of the editing complexes and their interactions with substrate RNAs. We propose to perform a high resolution structure- function analysis of the main component of the editing reaction - the 1 md RECC. We will attempt to confirm and extend our hypothetical editing reaction model by determining the 3D structure of these particles at an atomic resolution and identifying the protein components and their protein-protein and protein-RNA interactions in different functional states of the editing reaction The molecular structure information will be interpreted in terms of the detailed mechanism of this pathway and this knowledge will provide a basis for future investigations of the holoenzyme and the possible interactions of editing with mitochondrial RNA regulation and protein translation. This is important since the trypanosomatids have become important eukaryotic model systems for studying many basic biological phenomena in addition to RNA editing, such as protein expression, trans-splicing surface antigen variation and flagellar motility. Since it now appears that mitochondrial RNA editing may interact with translation and RNA regulation, an understanding of editing will assist in the development of trypanosomatids as model biological systems. In addition, U-insertion/deletion editing serves as a paradigm for multiple other post-transcriptional RNA modification phenomena such as fold-back guided A to I editing in human brain, sno-RNA guided methylation and pseudouridylation of ribosomal RNAs and even Si RNA-guided cleavage of mRNAs in RNAi, and the information obtained in this project may prove relevant for understanding these other biologically important phenomena. And last but not least, the editing pathway is essential for viability and may provide a potential target for development of interventions against the diseases caused by these parasites.