Molecular Mechanism of Chloroplast RNA Editing

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RNA editing is a form of RNA processing that occurs in diverse organisms. Editing is often essential for proper gene function and/or regulation. In vascular plants, mitochondrial and chloroplast RNAs are modified by C-to-U base modification. Editing sometimes creates start codons for plant organelle proteins, and is required to restore codons that specify highly conserved amino acids. RNA editing in plant organelles appears to be a correction mechanism to ameliorate T-to-C mutations that would otherwise prevent gene expression or result in non-functional proteins. Sequences surrounding C targets of RNA editing in chloroplasts are thought to be specifically recognized by proteins, perhaps recruiting other proteins involved in the enzymatic modification of C to U. The goal of this project is to identify unknown components of the chloroplast RNA editing apparatus. While cis-elements upstream of edited Cs have been identified, little is known about the trans-factors required to recognize the correct C and convert it to U. Efforts will be made to fractionate maize chloroplast extracts for partial purification of the editing apparatus, which will be subjected to mass spectrometry for protein identification. Candidate editing complex members will undergo functional analysis. A high-throughput assay for editing of all chloroplast editing sites in a species will be developed. RNA sequence requirements for binding of editing factors will be determined. Antisera to an editing factor protein will be used in purification schemes to identify components of the editing complex. A combination of biochemical and molecular genetic approaches should allow dissection of the molecular apparatus needed to convert Cs to Us in vascular plant chloroplasts. RNA editing is a fundamental biological process with profound effects on gene expression. Understanding how organelles in plants regulate this process may eventually result in applications that could improve agricultural productivity by increasing efficiency of energy acquisition and utilization by chloroplasts and mitochondria. This project will provide training in biochemistry, molecular biology, and genetics for several graduate students and undergraduates. Materials useful for laboratory exercises in plant cell structure and microscopy will be produced.