Structures and Functions of RNA Editing TUTases

描述(申请人提供)：锥虫是一种寄生的原生动物血鞭毛虫，在发展中国家造成健康问题。这些生物在进化早期就与其他真核生物不同，拥有许多独特的RNA处理途径，如尿苷插入/缺失、线粒体mRNAs编辑等。对RNA编辑和引导RNA成熟过程的研究强调，RNA尿苷基化反应是布鲁氏锥虫线粒体RNA生物发生的关键。我们发现了三种末端尿酰转移酶(TUTase)，它们具有独特的结构和基本功能。这一建议侧重于：1)RET1催化的3‘-尿苷基化在处理RNA前体中的功能；2)RET2引导U-插入的机制；以及3)MEAT1的生物学作用。我们认为这项研究对于开发TUTase抑制剂作为潜在的胰酶抑制剂是必不可少的。其具体目的是：1.研究RET1催化的小核糖体RNA 3‘-尿苷基化的功能。编辑由转录后被3‘U-加成修饰的反式作用引导RNA指导。在普遍存在的类gRNA分子和rRNA中也发现了类似的U-尾。我们认为，尿苷基化稳定了gRNA样分子，从而引导了上环和小环编码的多顺反子转录本的核裂解。我们将通过下一代测序和生化方法来分析短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。我们认为，U-插入编辑的保真度是由RET2的S对UTP和RNA底物的内在选择性决定的，而复杂的缔合有助于编辑效率。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.确定MEAT1 TUTase的功能。MEAT1是一种独有的U-特异性TUTase，它与20s编辑体样颗粒结合，对寄生虫的生存至关重要。我们建议调查U-插入编辑是否通过不同的RET2和MEAT1依赖机制完成。MEAT1特异的U-插入编辑位点和相互作用的伙伴将通过体内交联物和定量质谱学来鉴定。 公共卫生相关性：锥虫是发展中国家寄生虫病的病原体，包括美国大量存在的地区。现有的治疗方法往往是有毒和无效的，这进一步强调了新药的必要性。靶向基本的寄生虫特异性酶，如线粒体RNA编辑末端尿苷酰转移酶(TUTase)，是开发新一代抗锥虫药物的一条很有前途的途径。

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.