Structures and Functions of RNA Editing TUTases

描述（由申请方提供）：锥虫是寄生原生动物血鞭毛虫，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不同，具有许多独特的RNA加工途径，如线粒体mRNA的尿苷插入/缺失编辑。RNA编辑和指导RNA成熟过程的研究强调RNA尿苷酰化反应是布氏锥虫线粒体中RNA生物合成的关键。我们发现了三个末端尿苷酰转移酶（TUTases），独特的结构和基本功能的酶。这项建议的重点是：1）RET 1催化的3' -尿苷酰化在加工RNA前体中的功能; 2）RET 2引导U-插入的机制;和3）MEAT 1的生物学作用。我们认为这项研究是必不可少的TUTase抑制剂作为潜在的锥虫的发展。具体目标是：1。研究RET 1催化的小RNA和核糖体RNA的3' -尿苷酰化的功能。编辑由通过3' U -添加进行转录后修饰的反式作用向导RNA指导。在普遍存在的gRNA样分子和rRNA中也发现了类似的U尾。我们提出，尿苷酰化稳定gRNA样分子，其指导大环和小环编码的多顺反子转录物的溶核裂解。我们将通过下一代测序和生物化学方法分析短RNA的功能，序列多样性和稳定性。2.确定RET 2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RET 2对UTP和RNA底物的内在选择性决定的，而复杂的缔合有利于编辑效率。基于结构的预测将通过一种新的基于RNAi的诱导型基因敲入系统进行测试。3.建立MEAT 1 TUTase的功能。MEAT 1是一种专门的U特异性TUTase，与20 S编辑体样颗粒相关，对寄生虫的生存能力至关重要。我们建议调查是否U-插入编辑是由不同的RET 2和MEAT 1依赖的机制。将通过体内交联和定量质谱法鉴定MEAT 1特异性U插入编辑位点和相互作用伴侣。

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.