The role of non-canonical base pairs in RNA editing

非规范碱基对在 RNA 编辑中的作用

• 批准号: 8373126
• 负责人: Blaine H. M. Mooers
• 金额: $ 37.71万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8373126
• 关键词: 3-Dimensional; Address; Affect; African Trypanosomiasis; Area; Base Pair Mismatch; Base Pairing; Binding; Biological Assay; Biology; Calorimetry; Cessation of life; Chagas Disease; Cleaved cell; Complex; Data; Development; Double-Stranded RNA; Drug Delivery Systems; Drug Design; Goals; Guide RNA; Health; Human; In Vitro; Individual; Infection; Kinetics; Knowledge; Leishmania; Ligation; Major Groove; Measures; Messenger RNA; Mitochondria; Modeling; Nucleotides; Outcome; Parasites; Pharmaceutical Preparations; Point Mutation; Proteins; RNA; RNA Editing; RNA Folding; RNA Interference; Reaction; Resolution; Ribonucleoproteins; Roentgen Rays; Role; Site; Staining method; Stains; Structure; System; Testing; Therapeutic; Thermodynamics; Transcript; Trypanosoma; Variant; Width; X-Ray Crystallography; absorption; base; design; effective therapy; fighting; improved; inhibitor/antagonist; mRNA Precursor; research study; simulation; three dimensional structure

DESCRIPTION (provided by applicant): Twenty-four species of kinetoplastids (Trypanosoma and Leishmania) infect 30 million people worldwide and threaten 600 million people with debilitating and sometimes fatal infections. Kinetoplastids have a unique RNA editing system (kRNA editing) not found in humans, rendering a promising drug target. No one has effectively why non-canonical base pairing is a conserved feature of the binary mRNA-gRNA complex that is central to RNA editing in trypanosomes. Addressing this issue with structure-function- based studies is important because it is the first step in the rational design of drugs against the RNA editing complex. Our long-term goals are to understand the role of RNA structure in kRNA editing and more generally to relate sequence with structure. The immediate goal of this project is to determine how non-canonical RNA base pairs alter the structure and function of the U-helix domain of the pre- mRNA/gRNA complexes. Our central hypothesis is that non-canonical base pairs affect RNA editing by influencing the width of the major groove and by affecting the thermal stability of the double helix. The widening of the major groove may enhance recognition by proteins in the editosome and reduced thermal stability may allow absorption of torsional stain introduced by strand separation at the editing site before endonucleolytic cleavage of the pre-mRNA strand, and it may facilitate the progression of editing from the first editing site to th second editing site along the pre-mRNA strand. We test the hypothesis using structure-function studies of single and multiple point mutations in the U-helix. In aim 1, we test the effect of non-canonical base pairs near the editing sites in the template domain and in the U-helix domain on editing efficiency in vitro. In aim 2, we determine the effects of these non-canonical base pairs on the thermodynamic stability of the gRNA/mRNA duplex. In aim 3, we will determine the effects of non-canonical base pairs on the structure of the gRNA/mRNA duplex by X-ray crystallography. More accurate models of dsRNA-an expected outcome of this project-will contribute to design of inhibitors that target kRNA editing in trypanosomes but also impact other important areas such as 1) general design of dsRNAs including therapeutic RNAs, 2) more accurate predictions of RNA structure, and 3) simulations of RNA folding. The outcomes of this project are expected to advance in our understanding of the role of non-canonical base pairing in RNA editing. This advancement in understanding will aid the development of more effective therapies to fight infections with trypanosomes. PUBLIC HEALTH RELEVANCE: This project will determine the effect of RNA three-dimensional structure on the RNA editing complexes that are unique to the mitochondria of trypanosomes. The new knowledge gained from this project will provide critical data for the effort to improve the health of millions of people infected with these pathogenic parasites by developing safer and more effective treatments of trypanosome infections such as Chagas disease and sleeping sickness.

描述（由申请人提供）：二十四种动作塑料（锥虫瘤和利什曼尼亚）感染了全球3000万人，并威胁了6亿人患有衰弱的感染，有时甚至是致命的感染。动作塑料具有独特的RNA编辑系统（KRNA编辑），在人类中未发现，从而提供了有希望的药物靶标。没有人有效地为什么非规范基碱对是二元mRNA-GRNA复合物的保守特征，它是锥虫中RNA编辑的核心。通过基于结构功能的研究解决这个问题很重要，因为它是针对RNA编辑复合物的药物合理设计的第一步。我们的长期目标是了解RNA结构在KRNA编辑中的作用，而更普遍地将序列与结构相关联。该项目的直接目的是确定非规范的RNA碱基对如何改变前mRNA/GRNA复合物的U螺旋结构域的结构和功能。我们的中心假设是非规范基础对通过影响主要凹槽的宽度并影响双螺旋的热稳定性来影响RNA编辑。主要凹槽的扩大可能会增强蛋白质在编辑体中的识别和降低的热稳定性，可能会吸收在编辑位点上通过链分离引入的扭转染色，然后在核酸溶解位点进行核酸溶解裂解，并可以促进从第一次编辑位点进行编辑的沿二次编辑位点的进展，从而促进沿二次编辑位点的进展。我们使用U螺旋中的单点突变的结构 - 功能研究检验了假设。在AIM 1中，我们测试了模板域和U螺旋结构域中编辑位点附近的非规范基础对的影响，对体外编辑效率。在AIM 2中，我们确定了这些非规范基础对对GRNA/mRNA双链体热力学稳定性的影响。在AIM 3中，我们将通过X射线晶体学来确定非规范基础对对GRNA/mRNA双链体的结构的影响。 DSRNA的更准确的模型 - 该项目意愿的预期结果有助于靶向KRNA编辑的抑制剂的设计，但也影响了其他重要领域，例如1）DSRNA的一般设计，包括治疗性RNA，2）更准确的RNA结构预测，以及RNA折叠的模拟。预计该项目的结果将在我们理解非规范基础配对在RNA编辑中的作用方面发展。理解的进步将有助于开发更有效的疗法，以抗击锥虫感染。 公共卫生相关性：该项目将确定RNA三维结构对RNA编辑复合物的影响，该复合物是锥虫的线粒体所特有的。从该项目中获得的新知识将提供关键的数据，以努力通过开发更安全，更有效的锥虫感染（例如chagas疾病和睡眠疾病）来改善数百万人感染这些致病寄生虫的健康。