Biomedical subjects

Elaine M Byrne

Publications and source records attributed to Elaine M Byrne.

4 recordsLinked to original sources

PubMed2004

Unexpectedly complex editing patterns at dinucleotide insertion sites in Physarum mitochondria.

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.

Animals↗

PubMed2004

Chimeric templates and assays used to study physarum cotranscriptional insertional editing in vitro.

RNAs made in the mitochondrion of Physarum polycephalum are edited relative to their template by the precise addition of nonencoded nucleotides, while they are being synthesized. This insertional editing has been reproduced in vitro during run-on extension of RNAs initiated in vivo, within partially purified mitochondrial transcription elongation complexes (mtTECs), but it does not occur when the mitochondrial polymerase initiates transcription on exogenous cloned DNA. This chapter describes in vitro transcription systems in which mtTEC RNAs are elongated on repositioned parts of the genome or exogenous DNA, in order to investigate how the nontemplated insertions are directed. Restriction enzyme digestion and DNA ligation are used to generate the chimeric templates, and the RNA products are analyzed directly by nuclease dissection (S1 protection followed by RNase T1 digestion) or by reverse transcriptase-polymerase chain reaction (RT-PCR) followed by restriction enzyme analysis or cloning and sequencing.

Animals↗

PubMed2002

Editing site recognition and nucleotide insertion are separable processes in Physarum mitochondria.

Insertional RNA editing in Physarum polycephalum is a complex process involving the specific addition of non-templated nucleotides to nascent mitochondrial transcripts. Since all four ribonucleotides are substrates for the editing activity(s), both the site of insertion and the identity of the nucleotide to be added at a particular position must be specified, but the signals for these events have yet to be elucidated. Here we report the occurrence of sporadic errors in RNAs synthesized in vitro. These mistakes, which include omission of encoded nucleotides as well as misinsertions, occur only on templates that support editing. The pattern of these misediting events indicates that editing site recognition and nucleotide addition are separable events, and that the recognition step involves features of the mitochondrial template that are required for editing. The larger deletions lack all templated nucleotides between editing sites, suggesting that the transcription/editing apparatus can "jump" from one insertion site to another, perhaps mediated by interactions with editing determinants, while smaller omissions most likely reflect misalignment of the transcript upon resumption of templated RNA synthesis.

Animals↗

PubMed2002

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

RNAs in the mitochondrion of Physarum polycephalum are edited by the precise cotranscriptional addition of non-encoded 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.

Animals↗