Development of a cauliflower in vitro editing system and analysis of various cis-and-trans-factors

Most of the 400 RNA editing sites in flowering plant mitochondria are found in mRNAs. Consequently, the sequence vicinities ofhomologous sites are highly conserved between different species and are presumably recognized by likewise conserved trans-factors. To investigate the evolutionary adaptation to sequence variation, we have now analyzed the recognition elements of anediting site with divergent upstream sequences in the two species pea and cauliflower. This variation is tolerated at the siteselected, because the upstream cis-elements reach into the50-UTR of the mRNA. To compare cis-recognition features in pea andcauliflower mitochondria, we developed a new in vitro RNA editing system for cauliflower. In vitro editing assays with deletedand mutated template RNAs show that the major recognition elements for both species are located within the conservedsequence. In cauliflower, however, the essential upstream nucleotides extend further upstream than they do in pea. In-depthanalysis of single-nucleotide mutations reveals critical spacing of the editing site and the specific recognition elements, andshows that the +1 nucleotide identity is important in cauliflower, but not in pea.