Function of the Kinetoplast in the Hemoflagellates

描述（由申请人提供）：总体目标是在分子细节上了解在动着质体原生体线粒体中发生的称为尿苷插入/缺失RNA编辑的新型RNA修饰现象。最大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点插入和删除U来编辑，以产生可翻译的mrna。这种编辑涉及到转导rna的参与，它们决定精确的编辑位点，并调节要添加或删除的U的精确数量。我们的建议是鉴定所有参与这一过程的蛋白质和RNA，确定这些蛋白质是否需要通过RNA干扰下调表达来维持生存，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。在体外和体内重组编辑活性将使用重组蛋白进行。主要核心l -络合物的结构分析也将进行。突变l -复合物蛋白的功能互补分析也将在体外和体内进行，以确定特定保守基序的功能。RNA介导的l复合物和其他编辑复合物之间相互作用的性质将被研究。这项研究将为这些重要的人类和动物寄生虫的生存能力提供新的基因调控机制的宝贵信息。

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.