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Unexpectedly Complex Editing Patterns at Dinucleotide Insertion Sites in Physarum Mitochondria

Elaine M. Byrne and Jonatha M. Gott^*

Center for RNA Molecular Biology, School of Medicine, Case Western Reserve University, Cleveland, Ohio

Received 12 May 2004/ Returned for modification 1 June 2004/ Accepted 21 June 2004

Many of the RNAs transcribed from the mitochondrial genome of Physarum polycephalum are edited by the insertion of nonencoded nucleotides, which are added either singly or as dinucleotides. In addition, at least one mRNA is also subject to substitutional editing in which encoded C residues are changed to U residues posttranscriptionally. We have shown previously that the predominant type of editing in these organelles, the insertion of nonencoded single C residues, occurs cotranscriptionally at the growing end of the RNA chain. However, less is known about the timing of dinucleotide addition, and it has been suggested that these insertions occur at a later stage in RNA maturation. Here we examine the addition of both single nucleotides and dinucleotides into nascent RNAs synthesized in vitro and in vivo. The distribution of added nucleotides within individual cloned cDNAs supports the hypothesis that all insertion sites are processed at the same time relative to transcription. In addition, the patterns of partial editing and misediting observed within these nascent RNAs suggest that separate factors may be required at a subset of dinucleotide insertion sites and raise the possibility that in vivo, nucleotides may be added to RNA and then changed posttranscriptionally.

Present address: Centre for Bioengineering, Department of Mechanical Engineering, Trinity College, Dublin, Ireland.