Cotranscriptional editing of Physarum mitochondrial RNA requires local features of the native template

Abstract

RNAs in the mitochondrion of Physarum polycephalum are edited by the precise cotranscriptional addition of nonencoded nucleotides. Here we describe experiments to address the basis of editing specificity using a series of chimeric templates generated by either rearranging the DNA present in editing-competent mitochondrial transcription elongation complexes (mtTECs) or linking it to exogenous DNA. Notably, run-on transcripts synthesized from rearranged mtTECs are edited at the natural sites, even when different genes are ligated together, yet exogenous, deproteinized DNA does not support editing. Furthermore, the accuracy of nucleotide insertion in chimeric RNAs argues that any cis-acting determinants of cytidine insertion are limited to small regions surrounding editing sites. Taken together, these observations strongly suggest that template-associated factors affect read-out of the mitochondrial genome.

Unexpectedly Complex Editing Patterns at Dinucleotide Insertion Sites in Physarum Mitochondria

“…Of the nine clones which had not been substitutionally edited at the upstream C-to-U site (es30) and thus are known to be derived from nascent transcripts, five had a CA and four had a UA (Fig. 3B), while neighboring C insertion sites were fully and correctly edited, as expected on the basis of our previous results for atp mRNA (2). These data argue that at least a portion of the nascent RNA synthesized in vivo has CA rather than UA inserted at es31/32.…”

“…Each of these clones is clearly derived from nascent RNAs, as they contain a C residue at each of the four C-to-U sites. In addition, unlike the clones isolated from steady-state RNAs (7), none is fully edited, as expected for RNAs synthesized by mtTEC preparations (2,4). Of the 130 C insertion sites examined in this experiment, 56 (ϳ43%) were unedited, 67 (ϳ52%) were edited correctly, and 7 (ϳ5%) were misedited by the insertion of either a G or U residue or deletion of an adjacent encoded U residue.…”

“…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)(3)(4). 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.…”

“…Here we use an approach similar to that described above (2,3) 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.…”

Diversity and Evolution of Mitochondrial RNA Editing Systems

“…Because these three sub-types of insertion display unique evolutionary histories within the myxomycetes (i.e., the three do not always appear simultaneously in the same organism), it is conceivable they are mediated by distinct pathways (3,34). Development of an in vitro assay for Physarum insertional editing (35) has provided strong evidence that editing occurs co-rather than post-transcriptionally (4,36). Although the determinants of editing specificity in this system still have to be elucidated, at this point they do not appear to include gRNAs.…”