Function of the Kinetoplast in the Hemoflagellates

描述(申请人提供)：U-插入/缺失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。编辑通过在特定位置插入和删除尿氨酸来修改16个线粒体mRNAs中的12个的序列，这些mRNAs是从线粒体DNA“隐蔽基因”转录而来的，使它们可以翻译。我们发现了一类新的短RNA(引导RAS或gRNAs)，它包含通过反式碱基配对进行编辑的序列信息，使这种类型的RNA修饰符合中心教条。我们提出的基于gRNAs中介的假想编辑模型涉及多个3‘-5’循环，包括切割、U-添加或U-缺失和连接。我们为这个模型提出的负责酶活性的蛋白质被鉴定为1mD多蛋白复合体的组成部分，即RNA编辑核心复合体或RECC。另外几个可能参与RNA调控的多蛋白复合体通过RNA连接子与RECC相互作用。编辑发生在动质体谱系的所有成员中，包括锥虫寄生虫，这是几种人类疾病的病原体。如果不了解编辑复合体的分子结构及其与底物RNA的相互作用，就不能继续理解编辑。我们建议对编辑反应的主要成分-1MD RECC进行高分辨率结构-功能分析。我们将试图通过在原子分辨率下确定这些颗粒的三维结构并确定编辑反应不同功能状态下的蛋白质组分及其蛋白质-蛋白质和蛋白质-RNA相互作用来验证和扩展我们的假设编辑反应模型。分子结构信息将根据这一途径的详细机制进行解释，这些知识将为未来全酶的研究以及编辑与线粒体RNA调控和蛋白质翻译的可能相互作用提供基础。这一点很重要，因为锥虫已经成为除RNA编辑外研究许多基本生物学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，对编辑的理解将有助于锥虫作为模型生物系统的发展。此外，U-插入/缺失编辑可以作为多种其他转录后RNA修饰现象的范例，例如在人脑中折叠引导的A到I编辑，sno-RNA引导的核糖体RNA的甲基化和假尿基化，甚至在RNAi中由Si RNA引导的mRNAs切割，本项目中获得的信息可能被证明与理解这些其他重要的生物现象有关。最后但并非最不重要的一点是，编辑途径对于生存是必不可少的，并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的靶点。

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.