Exploring the Diversity of Plant RNA Editosomes

植物已经进化出一种RNA编辑机制，以防止发生在叶绿体和线粒体中基因突变的有害影响，这是光合作用和呼吸所必需的亚细胞细胞器。最近的研究表明，有不同的分子机器，RNA编辑体，执行这一重要的过程。并非所有RNA编辑设备都含有相同的蛋白质。为了了解RNA编辑机制是如何工作的，RNA编辑体的蛋白质组成将进一步利用遗传和生化方法进行表征。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使生物技术产业和社会受益。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，该项目涉及很大比例的未被充分代表的少数民族和来自其他学院的女性。除了科学方法外，学员还将在口头和书面报告实验数据方面得到指导。PI和coPI将继续为科学界提供行政服务。她将继续努力促进女性参与STEM领域。PI将发布一种新的多路质谱协议，可用于研究任何生物体中RNA编辑的个人。最近的进展导致对植物RNA编辑装置组成的了解大大增加，但任何特定编辑体的精确蛋白质组成尚未确定。除了PPR蛋白外，编辑体还包含另外三个蛋白家族（RIP、ORRM和OZ家族）的一个或多个成员，可能还有其他蛋白。拟议的实验将导致特定类型的RNA编辑体的详细表征。功能表位标记的OZ1和PPR蛋白版本将用于亲和纯化，以确定包含OZ1或PPR蛋白的编辑体的蛋白质组成。编码通过质谱分析鉴定的候选蛋白的基因将进行遗传分析，以确定它们是否为叶绿体RNA编辑所必需。将编辑事件转移到异源系统的可行性进行探讨。将获得的信息对于利用RNA编辑现象进行转录后水平的基因工程至关重要。本项目由分子和细胞生物科学部遗传机制项目资助。

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.