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The role of non-canonical base pairs in RNA editing

非规范碱基对在 RNA 编辑中的作用

基本信息

批准号：
8868892
负责人：
Blaine H. M. Mooers
金额：
$ 36.48万
依托单位：
UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8868892
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 Design Drug Targeting 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

项目摘要

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.

描述（由申请人提供）：24种着丝质体（锥虫和利什曼原虫）感染了全世界3000万人，并使6亿人受到致残，有时甚至致命的感染。着丝质体具有独特的RNA编辑系统（kRNA编辑），在人类中没有发现，提供了一个有希望的药物靶点。没有人有效地解释为什么非规范碱基配对是二元mRNA-gRNA复合体的保守特征，而二元mRNA-gRNA复合体是锥虫RNA编辑的核心。以结构-功能为基础的研究来解决这个问题是很重要的，因为这是合理设计抗RNA编辑复合物药物的第一步。我们的长期目标是了解RNA结构在kRNA编辑中的作用，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主槽的宽度和影响双螺旋的热稳定性来影响RNA编辑。主凹槽的加宽可以增强编辑体中蛋白质的识别能力，降低的热稳定性可能允许在前mrna链发生核内溶分裂之前，在编辑位点吸收由链分离引入的扭转染色，并可能促进沿前mrna链从第一个编辑位点到第二个编辑位点的编辑进展。我们使用u型螺旋单点和多点突变的结构-功能研究来检验这一假设。在目的1中，我们在体外测试了模板域和u -螺旋域编辑位点附近的非规范碱基对对编辑效率的影响。在目标2中，我们确定了这些非规范碱基对对gRNA/mRNA双工的热力学稳定性的影响。在目标3中，我们将通过x射线晶体学确定非规范碱基对对gRNA/mRNA双链结构的影响。更精确的dsrna模型-这个项目的预期结果-将有助于设计针对锥虫中kRNA编辑的抑制剂，但也会影响其他重要领域，如1)dsrna的一般设计，包括治疗性RNA， 2)更准确的RNA结构预测，以及3)RNA折叠模拟。该项目的结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。这一认识的进步将有助于开发更有效的治疗方法来对抗锥虫感染。