Structures and Functions of RNA Editing TUTases

描述（由申请人提供）：锥虫是一种寄生的原生动物血鞭毛虫，在发展中国家引起健康问题。这些生物在进化早期与其他真核生物分道扬镳，具有许多独特的RNA加工途径，如线粒体mrna的尿苷插入/删除编辑。RNA编辑和引导RNA成熟过程的研究强调，RNA尿苷化反应对布鲁氏锥虫线粒体中的RNA生物发生至关重要。我们发现了三种末端尿酰转移酶（TUTases），它们具有独特的结构和基本的功能。本研究的重点是：1)ret1催化的3' -尿苷化在RNA前体加工中的作用；2) RET2引导u插入的机制；3) MEAT1的生物学作用。我们认为这项研究对于开发TUTase抑制剂作为潜在的锥虫抑制剂是必不可少的。具体目标是：1。研究ret1催化的小rna和核糖体rna的3' -尿苷化的功能。编辑是由经过3' U添加的转录后修饰的反式引导rna指导的。类似的u型尾巴也存在于无处不在的类grna分子和rrna中。我们提出尿苷化可以稳定grna样分子，从而指导大环和小环编码的多顺反电子转录物的核裂解。我们将通过下一代测序和生化方法分析短rna的功能、序列多样性和稳定性。2. 确定ret2介导的u插入编辑反应的机制。我们提出，u插入编辑的保真度是由RET2对UTP和RNA底物的内在选择性决定的，而复杂的关联促进了编辑效率。基于结构的预测将通过一种新的基于rnai的可诱导基因敲入系统进行测试。3. 建立MEAT1 TUTase的功能。MEAT1是一种u -特异性TUTase，与20S编辑体样颗粒相结合，对寄生虫的生存至关重要。我们建议研究u插入编辑是否通过不同的RET2-和meat1依赖机制来完成。meat1特异性u插入编辑位点和相互作用伙伴将通过体内交联和定量质谱法鉴定。

DESCRIPTION (provided by applicant): Trypanosomes are parasitic protozoan hemoflagellates that cause health problems in developing countries. These organisms diverged from other eukaryotes early in evolution and possess many unique RNA processing pathways such as uridine insertion/deletion editing of mitochondrial mRNAs. Studies of the RNA editing and guide RNA maturation processes emphasize RNA uridylylation reactions as crucial for RNA biogenesis in mitochondria of Trypanosoma brucei. We discovered three Terminal Uridylyl Transferases (TUTases), enzymes of unique structures and essential functions. This proposal focuses on: 1) functions of the RET1-catalyzed 3' -uridylylation in processing RNA precursors; 2) the mechanism by which RET2 guides U-insertion; and 3) the biological role of MEAT1. We consider this research to be indispensable for the development of TUTase inhibitors as potential trypanocides. The Specific Aims are: 1. Investigate functions of RET1-catalyzed 3' -uridylylation of small and ribosomal RNAs. The editing is directed by trans-acting guide RNAs which are post-transcriptionally modified by the 3' U -addition. Similar U-tails are also found in ubiquitous gRNA-like molecules and in rRNAs. We propose that uridylylation stabilizes gRNA-like molecules, which direct nucleolytic cleavage of maxicircle- and minicircle-encoded multicistronic transcripts. We will analyze functions, sequence diversity, and stability of short RNAs by next-generation sequencing and biochemical methods. 2. Determine the mechanism of the RET2-mediated U-insertion editing reaction. We propose that the fidelity of the U-insertion editing is determined by RET2's intrinsic selectivity for UTP and RNA substrates while complex association facilitates the editing efficiency. Structure-based predictions will be tested by a novel RNAi-based inducible genetic knock-in system. 3. Establish the function of MEAT1 TUTase. MEAT1 is an exclusively U-specific TUTase which associates with a 20S editosome-like particle and is essential for the parasite's viability. We propose to investigate whether U-insertion editing is accomplished by distinct RET2- and MEAT1-dependent mechanisms. MEAT1-specific U-insertion editing sites and interacting partners will be identified by in vivo crosslinking and quantitative mass spectrometry. PUBLIC HEALTH RELEVANCE: Trypanosomatids are the causative agents of parasitic diseases in developing countries, including areas of substantial American presence. Available treatments are often toxic and ineffective, which further stresses the need for new drugs. Targeting essential parasite-specific enzymes, such as mitochondrial RNA editing terminal uridylyl transferases (TUTases), is a promising approach toward a new generation of trypanocides.