Function of the Kinetoplast in the Hemoflagellates

描述(由申请人提供)：总体目标是详细了解发生在动体原生生物线粒体中的称为尿苷插入/缺失RNA编辑的新的RNA修饰现象。上环线粒体基因组的信使RNA转录本必须在转录后通过在多个位置插入和缺失U‘s来编辑，以产生可翻译的mRNAs。这种编辑涉及反式作用的引导RNA的参与，它们确定准确的编辑位置，并调节要添加或删除的U的准确数量。我们的建议是鉴定参与这一过程的所有蛋白质和RNA，通过RNA干扰下调表达来确定这些蛋白质是否是生存所必需的，并在几个细胞系统中表达这些蛋白质，并分析它们的酶或结构特性。体外和体内编辑活动的重建将使用重组蛋白进行。还将对主要核心L建筑群进行结构分析。此外，还将在体外和体内对突变的L复合蛋白进行功能互补分析，以确定特定保守基序的功能。L-复合体和其他编辑复合体之间的相互作用的性质将被调查。这项研究应该会为这些重要的人类和动物寄生虫的生存所需的一种新的基因调控机制提供有价值的信息。

DESCRIPTION (provided by the applicant): The overall goal is to understand in molecular detail the novel RNA modification phenomenon known as uridine insertion/deletion RNA editing that occurs in the mitochondria of kinetoplastid protists. Messenger RNA transcripts of the maxicircle mitochondrial genome must be edited after transcription by insertion and deletion of U's at multiple sites to produce translatable mRNAs. This editing involves the participation of trans- acting guide RNAs which determine the precise editing sites and mediate the precise number of U's to be added or deleted. The proposal is to identify all of the proteins and RNAs involved in this process, to determine if these proteins are required for viability by RNA interference down regulation of expression, and to express the proteins in several cell systems and analyze their enzymatic or structural properties. Reconstitution of editing activities in vitro and in vivo will be performed using recombinant proteins. Structural analysis of the major core L-complex will also be performed. A functional complementation analysis of mutated L-complex proteins will also be performed in vitro and in vivo to determine the function of specific conserved motifs. The nature of the RNA mediated interactions between the L-complex and other editing complexes will be investigated. This research should yield valuable information on a novel gene regulation mechanism that is required for viability of these important human and animal parasites.