C to U editing and modifications during the maturation of the mitochondrial tRNA(Asp) in marsupials

In marsupial mitochondria, the nucleotide residue at the second position of the anticodon of the tRNA for aspartic acid is changed post-transcriptionally such that the translational machinery recognizes it as a uracil rather than the cytosine residue encoded in the gene. By postlabeling nucleotide analysis, we show here that the cytosine residue is converted to a conventional uracil residue in an RNA editing event that affects approximately half of the tRNA molecules under steady state conditions. Furthermore, we have identified three different tRNA(Asp) species which all carry three pseudouridines and two methylations but have the anticodons GCC, GUC and QUC respectively, the latter representing a rare example of queuine incorporation into a mitochondrial tRNA. This allows us to describe a likely sequential order of modification of the tRNA(Asp), where methylations and conversions of uridines to pseudouridines precede the editing event, while the exchange of guanine by queuine takes place after the C to U editing event.