The sequence content of many RNAs in a variety of organisms is completed or diversified by RNA editing, involving the substitution of bases or insertion and deletion of nucleotides during or after transcription. Extensive insertional editing occurs during transcription in Physarum
mitochondria, adding hundreds of single C residues, at an average spacing of approximately 25 nucleotides in mRNAs and 40 nucleotides in tRNAs and rRNAs, and a much smaller number of single U residues (6
). In addition, there are 19 reported instances of pairs of RNA residues known or likely to be inserted adjacent to each other (6
mitochondria are distinctive in also performing substitutional editing on a transcript that is insertionally edited: C-to-U conversion occurs at four known sites in the coI
mRNA, apparently at a later stage in RNA production (7
). Together, these editing events result in the creation of open reading frames encoding proteins involved in oxidative phosphorylation and electron transport, as well as the generation of mature mitochondrial tRNAs and rRNAs.
The factors responsible for single-nucleotide and dinucleotide insertions have yet to be identified. Phylogenetic comparisons indicate that the ability to insert single C nucleotides, single U nucleotides, and dinucleotides arose separately. Horton and Landweber (9
) determined the sequence of the mitochondrial coI
gene and its transcript among a collection of organisms with various degrees of relatedness to Physarum
. On the basis of their analysis, they proposed that U insertion appeared first, followed by C insertion and later dinucleotide additions, perhaps upon the emergence of separate or additional specificity factors (9
). It has also been proposed that single and dinucleotide insertions occur via distinct mechanisms in Physarum
). This suggestion was based on the results of experiments in which cytb
cDNAs containing unedited and misedited sites were selectively amplified from total mitochondrial RNA pools. Thus, there are a number of possible scenarios regarding the relationship between dinucleotide and mononucleotide insertional editing in Physarum
mitochondria. Dinucleotides might be inserted by separate activities which evolved independently or were derived from the mononucleotide insertion apparatus or by the same machinery, potentially with additional or alternative specificity factors.
The insertion of single C nucleotides occurs cotranscriptionally, with the extra nucleotides added to the growing 3′ end of the RNA (5
). Three lines of evidence indicate that dinucleotides are also added to nascent transcripts. First, S1 protection studies of labeled run-on transcripts synthesized in isolated mitochondria show that coI
mRNAs synthesized in vitro contain added nucleotides at the GU insertion site (13
). Second, pulse-chase studies indicate that nascent coI
transcripts associated with stalled RNA polymerases contain added nucleotides at the GU site (15
). Third, RNA fingerprint analyses indicate that the CU site within the coI
mRNA is also edited correctly in transcripts labeled in isolated mitochondria (14
). Thus, if a separate activity were responsible for processing of dinucleotide insertion sites, it would have to act shortly after C insertion, unlike the activity which generates the C-to-U substitutions in the same transcript (13
Our recent analyses of partially edited (2
) and misedited (3
) transcripts produced during run-on synthesis in mitochondrial transcription elongation complexes (mtTECs) have been informative in regards to the steps required for the insertion of C nucleotides. In these experiments, run-on transcription was performed in partially purified mtTECs, which contain mitochondrial genomes with associated RNA polymerases and nascent transcripts, and individual RNA molecules were characterized by reverse transcription-PCR (RT-PCR), cloning, and sequencing. In this system, the RNA synthesized in vitro is partially edited at C insertion sites, with edited, unedited, and occasional misedited sites interspersed along individual molecules (2
). This contrasts with RNA synthesized in vivo, which is efficiently and accurately edited at these sites (3
); thus, sequences downstream of unedited and misedited sites in run-on transcripts are known to have been extended in vitro. Interestingly, both the addition of only 1 nucleotide at dinucleotide insertion sites (14
) and misinsertion of a second residue at C insertion sites (3
) have been observed under certain conditions in vitro. These observations, taken together with the biochemical data of Visomirski-Robic and Gott (13
), led us to investigate whether single nucleotides and dinucleotides may be added at the same time relative to transcription, potentially by the same basic machinery.
Here we use an approach similar to that described above (2
) to examine further the relationship between dinucleotide and mononucleotide insertion, providing the first characterization of the sequences of individual nascent RNA molecules containing dinucleotide insertion sites. An interspersion of edited and unedited single and dinucleotide sites is observed, with rather complex patterns of addition at a subset of the dinucleotide sites. In addition, our data raise the intriguing possibility that in vivo, nucleotides may be added to RNA and then changed posttranscriptionally.