Addition of uridines to edited RNAs in trypanosome mitochondria occurs independently of transcription

Abstract

RNA editing is a novel RNA processing event of unknown mechanism that results in the introduction of nucleotides not encoded in the DNA into specific RNA molecules. We have examined the post-transcriptional addition of nucleotides into the mitochondrial RNA of Trypanosoma brucei. Utilizing an isolated organelle system we have determined that addition of uridines to edited RNAs does not require ongoing transcription. Trypanosome mitochondria incorporate CTP, ATP, and UTP into RNA in the absence of transcription. GTP is incorporated into RNA only as a result of the transcription process. Post-transcriptional CTP and ATP incorporation can be ascribed to known enzymatic activities. CTP is incorporated into tRNAs as a result of synthesis or turnover of their 3' CCA sequences. ATP is incorporated into the 3' CCA of tRNAs and into mitochondrial messenger RNAs due to polyadenylation. In the absence of transcription, UTP is incorporated into transcripts known to undergo editing, and the degree of UTP incorporation is consistent with the degree of editing occurring in these transcripts. Cytochrome b mRNAs, which contain a single editing site near their 5' ends, are initially transcribed unedited at that site. Post-transcriptional labeling of cytochrome b mRNAs in the organelle with [alpha-32P]UTP results in the addition of uridines near the 5' end of the RNA but not in a 3' region which lacks an editing site. These results indicate that RNA editing is a post-transcriptional process in the mitochondria of trypanosomes.