Regulation of RNA editing in Trypanosoma brucei

描述(申请人提供)：RNA编辑是锥虫体内一种新的和基本的基因调控机制，它通过特定的尿苷插入和缺失改变线粒体RNA，形成功能性的mRNAs。编辑的遗传信息通过碱基配对相互作用从Frans作用的gRNAs转移到mRNAs。虽然在了解核心编辑机制的结构和组成方面取得了相当大的进展，但影响编辑过程的准确性和效率的监管因素仍然难以捉摸。我们的实验室最近为第一个RNA编辑调节因子--RNA结合蛋白RBP16提供了遗传学证据。RNAi介导的RBP16在原环型(PF)锥体中的敲除导致细胞色素b(Cyb)RNA编辑的显著和特异的减少。在RBP16基因敲除中，Cyb gRNA的丰度没有受到影响，这表明调控发生在gRNA利用的水平上。与其在体内的作用一致，RBP16在体外可重复刺激RNA编辑高达5倍。初步数据表明，RBP16通过一种有趣的两步机制增强编辑，其中一个方面独立于特定的、高亲和力的RBP16-RNA相互作用。我们的目标是确定 RBP16 RNA编辑调控。在目标1中，我们将下调RBP16在血流形式(BF)锥体中的表达，并分析其对BF特异性和结构性编辑事件的影响。我们将通过分析PF和BF RBP16敲除细胞中与编辑体相关的gRNA来检验RBP16促进特定gRNAs招募到编辑体的假设。在目标2中，我们将直接测试RBP16刺激mRNA切割前和编辑后步骤的模型。通过体外实验比较突变型和野生型RBP16的RNP1和RGG RNA结合域在这一过程中的作用。在目标3中，将通过TAP标记的RBP16在PF和BF布氏锥虫中的表达和酵母双杂交筛选来鉴定与RBP16相互作用的蛋白。这种遗传和生化相结合的方法将为RNA编辑调控这一基本上未被探索的领域提供重要的洞察力。

DESCRIPTION (provided by applicant): RNA editing is a novel and essential gene regulatory mechanism in trypanosomes that alters mitochondrial RNAs by specific uridine insertion and deletion to form functional mRNAs. The genetic information for editing is transferred from frans-acting gRNAs to mRNAs through base-pairing interactions. While considerable progress has been made in understanding the structure and composition of the core editing machinery, regulatory factors that affect the accuracy and efficiency of the editing process remain elusive. Our laboratory recently provided genetic evidence for the first RNA editing regulatory factor, the RNA binding protein, RBP16. RNAi-mediated knock-down of RBP16 in procyclic form (PF) trypanosomes leads to a dramatic and specific decrease in editing of apocytochrome b (CYb) RNA. CYb gRNA abundance is unaffected in RBP16 knock-downs, suggesting that regulation takes place at the level of gRNA utilization. Consistent with its role in vivo, RBP16 reproducibly stimulates RNA editing in vitro up to 5-fold. Preliminary data suggest that RBP16 enhances editing by an intriguing two-step mechanism, one aspect of which is independent of specific, high affinity RBP16-RNA interactions. Our goal is to determine the scope and mechanism of RBP16 RNA editing regulation. In Aim 1, we will down-regulate RBP16 expression in bloodstream form (BF) trypanosomes and analyze the effects on BF-specific and constitutive editing events. We will test the hypothesis that RBP16 facilitates recruitment of specific gRNAs to editosomes by analysis of editosome-associated gRNAs in PF and BF RBP16 knock-down cells. In Aim 2, we will directly test the model that RBP16 stimulates both pre- and post-mRNA cleavage steps of editing. The role the RNP1 and RGG RNA binding domains of RBP16 in this process will be examined by comparison of mutant and wild type RBP16 in the in vitro assay. In Aim 3, RBP16-interacting proteins will be identified by both expression of TAP-tagged RBP16 in PF and BF T. brucei and by a yeast two-hybrid screen. This combined genetic and biochemical approach will provide important insight into the largely unexplored area of RNA editing regulation.