Molecular Definition of Plant Organelle Editosomes

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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.