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）：24种动质体（锥虫属和利什曼原虫属）感染全球3000万人，并威胁6亿人患上使人衰弱甚至致命的感染。动质体具有在人类中未发现的独特的RNA编辑系统（kRNA编辑），从而呈现出有希望的药物靶标。没有人能有效地解释为什么非规范碱基配对是二进制mRNA-gRNA复合物的保守特征，而该复合物是锥虫中RNA编辑的核心。通过基于结构-功能的研究来解决这个问题很重要，因为这是合理设计针对RNA编辑复合物的药物的第一步。我们的长期目标是了解RNA结构在kRNA编辑中的作用，并更广泛地将序列与结构联系起来。该项目的直接目标是确定非规范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，非规范碱基对通过影响大沟的宽度和影响双螺旋的热稳定性来影响RNA编辑。大沟的加宽可以增强编辑体中蛋白质的识别，并且降低的热稳定性可以允许在前体mRNA链的内切核酸裂解之前吸收由编辑位点处的链分离引入的扭转染色，并且它可以促进编辑沿着前体mRNA链从第一编辑位点沿着到第二编辑位点的进展。我们使用U螺旋中单个和多个点突变的结构-功能研究来检验这一假设。在目的1中，我们测试了模板结构域和U-螺旋结构域中编辑位点附近的非规范碱基对对体外编辑效率的影响。在目标2中，我们确定这些非典型碱基对对gRNA/mRNA双链体的热力学稳定性的影响。在目标3中，我们将通过X射线晶体学确定非典型碱基对gRNA/mRNA双链体结构的影响。更准确的dsRNA模型-该项目的预期成果-将有助于设计靶向锥虫中kRNA编辑的抑制剂，但也影响其他重要领域，如1）dsRNA的一般设计，包括治疗性RNA，2）更准确的RNA结构预测，3）RNA折叠模拟。该项目的成果有望促进我们对非规范碱基配对在RNA编辑中的作用的理解。这种理解的进步将有助于开发更有效的治疗方法来对抗锥虫感染。 公共卫生关系：该项目将确定RNA三维结构对锥虫线粒体所特有的RNA编辑复合物的影响。从该项目中获得的新知识将为改善数百万感染这些致病寄生虫的人的健康提供关键数据，方法是开发更安全，更有效的锥虫感染治疗方法，如恰加斯病和昏睡病。