Insertional editing of mitochondrial tRNAs of Physarum polycephalum and Didymium nigripes.

RNA editing has been shown to be an obligatory step in the gene expression of a number of organisms (26). The most extensively edited RNAs identified to date are produced in mitochondria or chloroplasts. In trypanosome mitochondria, uridines are inserted or deleted in mRNAs (28), and in the mitochondria of the acellular slime mold Physarum polycephalum, cytidines (and occasionally other nucleotides) are inserted in mRNA (6, 13, 20–22) or rRNAs (14). In mitochondria and chloroplasts of plants, some cytidine and uridine residues in RNA are converted by nucleotide modification to uridine and cytidine, respectively (15). Nucleotide modification in tRNAs has been known for decades (3). Generally, this consists of posttranscriptional modification of existing nucleotides but in the case of the Q base modification may also include the exchange of bases between nucleotides by transglycosylation (23). Usually, conventional ribonucleotides are converted to unconventional ribonucleotides. In a few instances, a conventional nucleotide has been converted to one of the other conventional ribonucleotides, and this has been termed tRNA editing. At least five different types of tRNA editing have been identified, all in mitochondria. Cytidine-to-uridine conversion has been observed in mitochondrial tRNAPhe and tRNACys of angiosperms (2, 17) and mitochondrial tRNAHis of a gymnosperm (18). These editing sites are located in the acceptor or anticodon stems of the tRNA and create A · U base pairs. Another cytidine-to-uridine conversion has been identified in the anticodon of the tRNAAsp of marsupials (7). This conversion changes the 5′-GCC-3′ anticodon to 5′-GUC-3′, allowing the tRNA to recognize aspartic acid codons and to be acylated with aspartic acid (4). In the mitochondria of the protist Acanthamoeba castellanii (5, 11, 12) and the fungus Spizellomyces punctatus (10), base substitutions occur in the first three nucleotides at the 5′ end of the tRNAs, creating conventional base pairs in the acceptor stem. Generally, G or A is substituted for the U, A, or C inferred from the mitochondrial DNA (mtDNA) sequence. In the mitochondria of snails (32), squid (29), and chickens (34), the 3′ ends of some tRNAs have base substitutions relative to the mtDNA sequence. These tRNAs are produced from the upstream gene of overlapping tRNA genes and have A substitutions. These tRNAs are thought to be truncated by 5′ processing of the downstream overlapping tRNA with subsequent oligo(A) addition to the 3′ end, which is terminated by CCA addition to produce mature tRNAs with 1 to 5 A · U bp at the end of the acceptor stem. A similar type of base substitution occurs in mitochondrial tRNASer of the platypus, in which 3 of the 4 nucleotides at the 3′ end of the tRNA are substituted relative to the mtDNA sequence (33). However, in this case the substituted nucleotides are not all adenosines. Here we report the identification and characterization of insertional editing in tRNAs of P. polycephalum and Didymium nigripes. These tRNAs have nucleotide insertions of cytidine or uridine relative to the mtDNA genes encoding them. These insertions create classical base pairing or conserved nucleotides in the tRNAs that are not predicted from the DNA sequence. cDNAs corresponding to unprocessed polycistronic tRNAs generally lack many of the nucleotide insertions at the editing sites.