Regulation of RNA editing in Trypansoma brucei

RNA编辑是锥虫中一种新的和必要的基因调控机制， 线粒体！RNA通过特异性尿苷插入和缺失形成功能性mRNA。遗传 用于编辑的信息通过碱基配对从反式作用gRNA转移到mRNA 交互.虽然在了解结构和 核心编辑机制的构成、调控因素影响编辑的准确性和效率 编辑过程中仍然难以捉摸。我们的实验室最近提供了第一个 RNA编辑调节因子：RNA结合蛋白，RBP 16。RNAi介导的敲低 原环形式（PF）锥虫中的RBP 16导致在对DNA的编辑中的显著和特异性减少。 脱辅基细胞色素B（CYB）RNA。CYb gRNA丰度在RBP 16敲低中不受影响， 这表明调控发生在gRNA利用水平。与其在体内的作用一致， RBP 16在体外可重复地刺激RNA编辑高达5倍。初步数据显示， RBP 16通过一个有趣的两步机制增强编辑，其中一个方面是独立的， 特异性、高亲和力RBP 16-RNA相互作用。我们的目标是确定 RBP 16 RNA编辑调控。在目标1中，我们将下调血液中RBP 16的表达， 形成（BF）锥虫并分析对BF特异性和组成性编辑事件的影响。我们 将检验RBP 16通过以下方式促进特异性gRNA募集到编辑体的假设： PF和BF RBP 16敲低细胞中编辑体相关gRNA的分析。在目标2中，我们将 直接测试RBP 16刺激编辑的前和后mRNA切割步骤的模型。 RNP 16的RNP 1和RGG RNA结合结构域在这一过程中的作用将通过以下方法进行检查： 体外测定中突变体和野生型RBP 16的比较。在目标3中，RBP 16相互作用 通过TAP标记的RBP 16在PF和BFT 16中的表达来鉴定蛋白质。布鲁塞和由a 酵母双杂交筛选这种遗传和生物化学相结合的方法将提供重要的 深入了解RNA编辑调控的大部分未开发领域。

RNA editing is a novel and essential gene regulatory mechanism in trypanosomes that alters mitochondria! 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 factori'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-RNAinteractions. 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.