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.
