Assay of editing of exogenous RNAs in chloroplast extracts of Arabidopsis, maize, pea, and tobacco.

Nucleotides within transcripts of chloroplasts and mitochondria are modified through C-to-U RNA editing in vascular plants. The specific protein components and enzymatic machinery required for editing have not been defined. A consensus sequence is not present around all editing sites, complicating the discovery of cis-sequence elements critical for editing. Chloroplast extracts capable of carrying out editing in vitro along with precise quantification of editing extent of exogenous transcripts will facilitate identification of both cis and trans factors. We have optimized an in vitro assay originally developed to study editing in tobacco and pea chloroplasts and have expanded the assay to include the study of chloroplast editing in the model species Arabidopsis and the monocot maize. The superior genetic resources in these two species can now be utilized in conjunction with biochemical analysis to dissect the editing apparatus. We have improved the assay conditions for editing in vitro, achieving efficient editing (as much as 92%) with certain RNA substrates. Unlike the initial assay that relied on qualitative analysis, we are able to achieve precise quantification of editing activity within 1% through a simple poisoned primer extension (PPE) assay with radiolabeled oligonucleotides.