Regulation of RNA editing in Trypansoma brucei

RNA编辑是锥虫体内一种新的和基本的基因调节机制，它可以改变 线粒体！通过特定的尿苷插入和缺失来形成具有功能的RNA。基因 编辑信息通过碱基配对从Frans作用的gRNAs转移到mRNAs 互动。虽然在理解结构和结构方面取得了相当大的进展 核心编辑机构的组成、影响精确度和效率的监管因素 编辑过程的细节仍然难以捉摸。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，RBP16。RNAi介导的基因敲除 原环状(PF)锥体中的RBP16导致编辑的显著和特定的减少 凋亡素b(Cyb)RNA。Cyb gRNA丰度在RBP16基因敲除中不受影响， 这表明调控发生在gRNA利用的水平上。与它在体内的作用一致， RBP16可重复刺激体外RNA编辑，最高可达5倍。初步数据显示， RBP16通过有趣的两步机制增强了编辑，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在血液中的表达 形成(BF)锥体，并分析其对BF特有的和构成编辑事件的影响。我们 将检验RBP16通过以下方式促进特定gRNA招募到编辑体的假设 Pf和BF RBP16基因敲除细胞中编辑体相关基因的分析。在目标2中，我们将 直接测试RBP16刺激编辑前后的mRNA切割步骤的模型。 RBP16的RNP1和RGG RNA结合域在这一过程中的作用将通过以下方式进行研究 突变型和野生型RBP16的体外实验比较。在目标3中，RBP16--互动 蛋白质将通过在PF和BF布鲁氏锥虫中表达TAP标记的RBP16和通过A 酵母双杂交筛选。这种遗传和生化相结合的方法将提供重要的 对RNA编辑调控这一基本未被探索的领域的洞察。

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.