Identification of RNA editing factors in the primitive eukaryote, Physarum

原始真核生物绒泡菌中 RNA 编辑因子的鉴定

• 批准号: 1616411
• 负责人: Jonatha Gott
• 金额: $ 60万
• 依托单位: Case Western Reserve University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-15 至 2020-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1616411&HistoricalAwards=false
• 关键词: Identification; RNA; editing; factors; primitive

The goal of this research is to identify factors responsible for RNA editing. RNA editing involves specific alterations of RNA molecules, relative to the genes from which they were copied, and affects expression of genes in mitochondria, the organelles responsible for energy generation in the cell. Whereas, RNA editing is widespread in many organisms, including humans, the underlying mechanisms are not well understood. This research will fill that knowledge gap and provide new insights how RNA editing occurs. The project will also provide training opportunities at many levels through concerted efforts to involve high school, undergraduate, and graduate students. One such effort will be to continue ongoing work with a high school teacher and students to develop experiments that can be done in an inner city school setting. Specific outreach to women and minority students will help broaden participation in science. Students will take part directly in the research, gaining valuable experience by learning how to optimize methods, master state-of-the-art techniques, and present the results of their work as part of the project's dissemination of the research outcomes to the broader scientific community. RNA editing occurs in many organisms and has been most extensively studied in humans and other mammals, plants, and human pathogens. Observed sequence changes include base conversions, base substitutions, the deletion of encoded nucleotides, and the insertion of additional residues. The only organism known to carry out all of these forms of editing is the primitive eukaryote Physarum polycephalum. The sequence of virtually all of the mRNAs, rRNAs, and tRNAs encoded in Physarum mitochondria differs from that of the mitochondrial genome in highly specific and reproducible ways. Many RNAs contain so many non-encoded nucleotides that their genes escaped identification for years. Previous work has shown that "extra" nucleotides are added to these RNAs during their synthesis and that the specificity of editing depends on template elements that flank editing sites; however, the underlying mechanism is still mysterious. The major aim of this project is to identify the factors that are required for these site-specific changes to mitochondrial RNAs. Previous work on two different forms of editing has narrowed the list of potential candidates for each type and these will be investigated further. The expression of each of these proteins will be knocked down individually and mitochondrial RNAs will be analyzed to determine whether RNA editing levels are reduced. Once editing factors are identified, their interactions with other mitochondrial proteins will be examined. Long-term goals for the project include the development of an in vitro system assembled from defined components for use in mechanistic studies and the definition of the specific roles played by both template elements and auxiliary factors. Successful completion of these studies will significantly enhance current understanding of how RNA sequence changes can be targeted in an exquisitely specific manner.

这项研究的目标是确定负责RNA编辑的因素。RNA编辑涉及到RNA分子相对于复制它们的基因的特定变化，并影响线粒体中基因的表达，线粒体是负责细胞中产生能量的细胞器。然而，RNA编辑在包括人类在内的许多生物体中都很普遍，其潜在的机制尚不清楚。这项研究将填补这一知识空白，并为RNA编辑是如何发生的提供新的见解。该项目还将通过共同努力，让高中生、本科生和研究生参与进来，提供多个层面的培训机会。其中一项努力将是继续与一名高中教师和学生进行正在进行的工作，以开发可以在市中心的学校环境中进行的实验。针对妇女和少数族裔学生的具体接触将有助于扩大对科学的参与。学生将直接参与研究，通过学习如何优化方法，掌握最先进的技术，并将他们的工作成果作为项目向更广泛的科学界传播研究成果的一部分，获得宝贵的经验。RNA编辑存在于许多生物体中，在人类和其他哺乳动物、植物和人类病原体中得到了最广泛的研究。观察到的序列变化包括碱基转换、碱基替换、编码核苷酸的缺失和额外残基的插入。已知的唯一进行所有这些形式编辑的生物是原始的真核生物多头绒泡菌。绒泡菌线粒体中编码的几乎所有的mRNAs、rRNAs和tRNAs的序列与线粒体基因组的序列在高度特异性和重复性方面不同。许多RNA含有如此多的非编码核苷酸，以至于它们的基因多年来没有被识别出来。以前的工作表明，在合成这些RNA的过程中，会向它们添加“额外的”核苷酸，编辑的特异性取决于编辑位点两侧的模板元素；然而，潜在的机制仍然是神秘的。该项目的主要目的是确定线粒体RNA的这些特定部位的变化所需的因素。以前关于两种不同形式的编辑的工作已经缩小了每种类型的潜在候选者的名单，这些将被进一步调查。这些蛋白质的表达将被逐一下调，线粒体RNA将被分析，以确定RNA编辑水平是否降低。一旦确定了编辑因子，就会检查它们与其他线粒体蛋白的相互作用。该项目的长期目标包括开发一个由已定义的组件组装而成的体外系统，用于机械学研究，并确定模板元件和辅助因子所发挥的具体作用。这些研究的成功完成将大大增强目前对RNA序列变化如何以精细具体的方式进行靶向的理解。