Factors Involed in Chloroplast RNA Editing

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DESCRIPTION (provided by applicant) The long-term objective of this project is to define RNA:RNA and RNA:protein interactions involved in RNA editing. In humans, editing is critical for RNA processing in the expression of several disease-related genes including the Wilm?s tumor susceptibility gene, glutamate receptor subunit B, and apolipoprotein B. This research will elucidate the factors that direct cytidine to uridine editing and increase our understanding of organellar editing mechanisms. During the fellowship period, transgenic and biochemical techniques will be used to identify molecules that interact with the chloroplast rpoB-I editing site. To study the role of RNA secondary structure in editing, rpoB-I will be fused to antisense sequences and expressed in transgenic tobacco chloroplasts. Electrophoretic mobility-shift assays will be performed to identify chloroplast proteins with binding specificity for in vitro transcribed rpoB-I editing sites. The nature of the RNA:protein interaction will be examined using modified editing sequences. The purification and characterization of putative editing proteins will be facilitated by an affinity approach. A functional screen will be utilized for the isolation of genes encoding editing factors.