Molecular Definition of Plant Organelle Editosomes

陆地植物的基因组包含在三个位置：细胞核和细胞器，叶绿体和线粒体。一种新的机制已经发展到纠正细胞器基因组中的错误。在DNA转录成RNA之后。一种叫做“编辑体”的蛋白质复合体在C存在的位置将C变为U，而不是正确的U核苷酸。利用植物细胞器编辑系统可以提供一种改变任何选定转录本的技术。例如，Cs可以转化为Us，通过引入起始密码子或终止密码子来修饰基因表达，或者允许产生功能蛋白。因此，在这个项目中取得的进展可能会导致一种变革性技术的出现，这将有利于生物技术行业和社会。该项目还将对教育产生影响，为一名研究生和几名本科生提供研究培训。该项目包括与少数族裔服务机构加州州立大学洛杉矶分校的一名教员合作，并为那里的本科生提供现场或康奈尔大学参与项目的机会。了解植物细胞器编辑体（负责C到U转化的分子装置）的组成和功能，是释放这一分子机制巨大潜力的先决条件。遗传和生化证据表明，在编辑体的组成中有四个不同的蛋白质家族。一个非凡的rna结合蛋白家族，五肽重复（PPR）蛋白，通过与数百种不同的上游顺式元件结合，选择合适的C进行编辑。辅助编辑因子的三个小家族是在被子植物中发生编辑的绝对必要的。最近的一项突破导致了一种新的细菌表达系统，该系统允许在大肠杆菌中提供单个PPR蛋白和一个辅助因子后编辑植物RNA序列。本研究的目的是利用这种异源表达系统来分析PPR蛋白和辅助因子单独或联合在编辑效率和特异性方面的作用。该项目将确定辅助因子如何影响RNA结合亲和力，以及它们在单一类型编辑体中的化学计量。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Land plants contain genomes in three locations: the nucleus and the organelles, both chloroplast and mitochondria. A novel mechanism has evolved to correct errors in organelle genomes. Following transcription of DNA to RNA. a protein complex called the “editosome” changes the C to a U at locations where a C is present instead of the proper U nucleotide. Harnessing the plant organelle editing system could offer a technology to alter any selected transcript. For example, Cs could be converted to Us to modify gene expression by introducing start codons or stop codons or to allow production of a functional protein. Advances made during this project could therefore lead to the emergence of a transformative technology that would benefit the biotechnology industry and society. The project will also have educational impact by providing research training for a graduate student and several undergraduates. The project includes a collaboration with a faculty member at a minority-serving institution, California State Los Angeles, and opportunities for undergraduates there to participate in the project on-site or at Cornell. Understanding the composition and functioning of plant organelle editosomes, the molecular apparatus responsible for the C to U conversion, is a prerequisite to unleash the tremendous potential of this molecular machinery. Genetic and biochemical evidence have implicated four different families of proteins in the composition of the editosome. A remarkable family of RNA-binding proteins, the pentatricopeptide repeat (PPR) proteins, select the proper C to be edited by binding to hundreds of different upstream cis-elements. Three small families of accessory editing factors are absolutely necessary for editing to occur in angiosperms. A recent breakthrough resulted in a new bacterial expression system that allowed editing of a plant RNA sequence in E. coli following provision of a single PPR protein along with one accessory factor. The goal of this research is to use this heterologous expression system to dissect the role of the PPR protein and accessory factors, alone and in combination, with regard to efficiency and specificity of editing. This project will determine how accessory factors affect RNA binding affinity and in what stoichiometry they are present in single types of editosomes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.