Exploring the Diversity of Plant RNA Editosomes

植物已经进化出一种RNA编辑机制，以防止位于叶绿体和线粒体中的基因发生突变的有害影响，叶绿体和线粒体是光合作用和呼吸作用所必需的亚细胞器。最近的研究表明，有不同的分子机器，RNA编辑小体，执行这一重要过程。并不是每个RNA编辑装置都含有相同的蛋白质。为了了解RNA编辑机制是如何工作的，将使用遗传学和生物化学方法进一步表征RNA编辑小体的蛋白质组成。通过识别改变RNA序列所需的所有蛋白质，编辑可以在未来用于故意修改由基因产生的RNA产物和蛋白质。这种技术可以通过在许多不同领域的应用而造福于生物技术行业和社会。该项目将为研究生和本科生提供培训机会，包括参加NSF REU项目的学生，该项目涉及很高比例的代表不足的少数族裔和来自其他大学的女性。除了科学方法，受训者还将在口头和书面陈述他们的实验数据方面得到指导。科联和科联都将继续为科学界提供行政服务。国际妇女联合会将继续努力促进妇女参与STEM领域的工作。PI将发布一种新的多路复用质谱学协议，该协议可用于研究任何有机体中的RNA编辑的个人。最近的进展使人们对植物RNA编辑装置的组成有了更多的了解，但还没有确定任何特定编辑小体的准确蛋白质组成。除了PPR蛋白外，编辑体还包含三个额外的蛋白质家族的一个或多个成员，RIP、Orrm和OZ家族，可能还有额外的蛋白质。拟议的实验将导致对特定类型的RNA编辑小体的详细描述。OZ1和PPR蛋白的功能表位标记版本将用于亲和纯化，以确定包含OZ1或PPR蛋白的编辑小体的蛋白质。通过质谱分析确定的编码候选蛋白质的基因将接受遗传分析，以确定它们是否需要叶绿体RNA编辑。将探讨将编辑事件转移到异类系统的可行性。将获得的信息对于在转录后水平上利用RNA编辑现象进行基因工程是至关重要的。该项目由分子和细胞生物科学部遗传机制计划资助。

Plants have evolved an RNA editing mechanism to prevent the detrimental effects of mutations that have occurred in genes located in chloroplast and mitochondria, which are subcellular organelles necessary for photosynthesis and respiration. Recent studies indicate that there are diverse molecular machines, RNA editosomes,that carry out this important process. Not every RNA editing apparatus contains the same proteins. In order to understand how the RNA editing mechanism works, the protein composition of RNA editosomes will be further characterized using genetic and biochemical methods. By identifying all of the proteins needed for changing the RNA sequence, editing could be used in the future for deliberate modification of the RNA product and protein produced from a gene. Such technology could benefit the biotechnology industry and society through application in many different fields. The project will provide training opportunities for both graduate and undergraduate students, including students participating in NSF REU program that involves a high proportion of underrepresented minorities and women from other colleges. In addition to scientific approaches, trainees will be mentored in both oral and written presentation of their experimental data. Both the PI and coPI will continue to perform administrative service for the scientific community. The co-PI will continue her efforts to promote participation of women in STEM fields. The PI will distribute a new multiplexed mass spectrometric protocol that can be used for individuals studying RNA editing in any organism. Recent progress has resulted in much increased knowledge of the composition of the plant RNA editing apparatus, but the precise protein composition of any particular editosome has not been identified. In addition to a PPR protein, an editosome contains one or more members of three additional protein families, the RIP, ORRM, and OZ families and perhaps additional proteins. Proposed experiments will lead to detailed characterization of particular types of RNA editosomes. Functional epitope-tagged versions of OZ1 and a PPR protein will be used for affinity purification to determine the proteins comprising editosomes that contain either OZ1 or the PPR protein. Genes encoding candidate proteins identified by mass spectrometry analysis will be subjected to genetic analysis to determine whether they are required for chloroplast RNA editing. The feasibility of transfer of an editing event to a heterologous system will be explored. The information that will be gained is essential for exploiting the phenomenon of RNA editing for genetic engineering at the post-transcriptional level.This project is funded by the Genetic Mechanisms Program in the Division of Molecular and Cellular Biosciences.