Editing site recognition in plant mitochondria: The importance of 5'-flanking sequences

Cytidine to uridine (C-to-U) editing occurs in plant mitochondria with very high specificity such that only specific cytidines are converted to uridines. The mechanisms for editing site selection in plant mitochondria are unknown. In order to examine the determinants of editing site recognition, repeated mitochondrial DNA sequences that include edited nucleotides have been evaluated as editing substrates. During evolution the maize mitochondrial ribosomal protein subunit 12 (rps12) gene recombined with intron 1 of the ribosomal protein subunit 3 (rps3) gene and a region of the S1-like sequence of the 2.3 kb plasmid. These recombinations created a second copy of an internal portion of the rps12 gene, known as rps12b, which includes the first four editing sites of rps12 transcripts. The duplicated sequence extends seven nucleotides upstream of editing site 1 and six nucleotides downstream from editing site 4. The sequences of rps12 and rps12b are identical between these sites except for a single change at -5 from editing site 1. These modifications did not affect C-to-U conversion at editing sites 2, 3, or 4 in rps12b; however, no editing was detected at editing site 1 in rps12b cDNAs. Thus, the 5' recombination abolished editing at site 1, while the 3' recombination modified the downstream RNA sequence, but did not effect editing at site IV. Secondary structure prediction suggests that changes in editing site recognition do not correlate with differences in secondary structures, and that primary RNA sequence may be responsible for editing site specification.