The role of non-canonical base pairs in RNA editing

描述(申请人提供)：24种动殖体(锥虫和利什曼原虫)感染全球3000万人，并威胁6亿人的虚弱，有时甚至致命的感染。动质体有人类所没有的独特的RNA编辑系统(Krna编辑)，使其成为一个有希望的药物靶点。没有人有效地解释为什么非正则碱基配对是二元mRNA-gRNA复合体的一个保守特征，该复合体是锥虫RNA编辑的中心。通过基于结构功能的研究来解决这个问题是重要的，因为这是针对RNA编辑复合体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，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复合体的U-螺旋域的结构和功能。我们的中心假设是，非正则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。主槽的加宽可能增强编辑小体中蛋白质的识别，降低热稳定性可能允许编辑部位在前mRNA链内切之前吸收由链分离引入的扭转染色，并可能促进编辑从第一编辑部位向第二编辑部位的进展。我们通过对U-螺旋中单点和多点突变的结构-功能研究来验证这一假设。在目标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正则碱基对对体外编辑效率的影响。在目标2中，我们确定了这些非正则碱基对对gRNA/mRNA双链热力学稳定性的影响。在目标3中，我们将通过X射线结晶学来确定非正则碱基对对gRNA/mRNA双链结构的影响。更准确的dsRNA模型--这是该项目的预期结果--将有助于针对锥体中Krna编辑的抑制剂的设计，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折叠的模拟。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。这一认识上的进步将有助于开发更有效的治疗方法来对抗锥虫感染。

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.