Identification of critical nucleotide positions for plastid RNA editing site recognition

Transcripts in higher plant cell organelles undergo RNA editing by C-to-U conversion. Both the mechanistic steps and the factors involved in this process are largely unknown. To gain a better understanding of the molecular interactions involved in organellar RNA editing, we have begun to identify critical nucleotide positions for plastid RNA editing-site recognition. We performed a scanning point mutagenesis on a sequence motif separating editing sites IV and V in the tobacco ndhB transcript. The constructs were integrated into the chloroplast genome by the biolistic process and the effect of each point mutation on editing of both the upstream and the downstream site was measured. In addition to a previously identified sequence element located upstream of both sites, only few nucleotide positions 5' and 3' of an editing site turned out to be of critical importance. Unexpectedly, our study revealed that mutation of the upstream site leads to loss of editing at the downstream site. However, our results also indicate that, even though closely adjacent editing sites can share common recognition elements in cis, they are edited independently and not in a polar fashion.