Complete characterization of the edited transcriptome of the mitochondrion of Physarum polycephalum using deep sequencing of RNA

Department of Physics, Department of Biochemistry and Center for RNA Biology, Ohio State University, Columbus, OH 43210, USA.
Nucleic Acids Research (Impact Factor: 9.11). 04/2011; 39(14):6044-55. DOI: 10.1093/nar/gkr180
Source: PubMed


RNAs transcribed from the mitochondrial genome of Physarum polycephalum are heavily edited. The most prevalent editing event is the insertion of single Cs, with Us and dinucleotides also added
at specific sites. The existence of insertional editing makes gene identification difficult and localization of editing sites
has relied upon characterization of individual cDNAs. We have now determined the complete mitochondrial transcriptome of Physarum using Illumina deep sequencing of purified mitochondrial RNA. We report the first instances of A and G insertions and sites
of partial and extragenic editing in Physarum mitochondrial RNAs, as well as an additional 772 C, U and dinucleotide insertions. The notable lack of antisense RNAs in
our non-size selected, directional library argues strongly against an RNA-guided editing mechanism. Also of interest are our
findings that sites of C to U changes are unedited at a significantly higher frequency than insertional editing sites and
that substitutional editing of neighboring sites appears to be coupled. Finally, in addition to the characterization of RNAs
from 17 predicted genes, our data identified nine new mitochondrial genes, four of which encode proteins that do not resemble
other proteins in the database. Curiously, one of the latter mRNAs contains no editing sites.

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Available from: Janine Altmüller, Nov 12, 2014
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    ABSTRACT: RNA editing describes the process in which individual or short stretches of nucleotides in a messenger or structural RNA are inserted, deleted, or substituted. A high level of RNA editing has been observed in the mitochondrial genome of Physarum polycephalum. The most frequent editing type in Physarum is the insertion of individual Cs. RNA editing is extremely accurate in Physarum; however, little is known about its mechanism. Here, we demonstrate how analyzing two organisms from the Myxomycetes, namely Physarum polycephalum and Didymium iridis, allows us to test hypotheses about the editing mechanism that can not be tested from a single organism alone. First, we show that using the recently determined full transcriptome information of Physarum dramatically improves the accuracy of computational editing site prediction in Didymium. We use this approach to predict genes in the mitochondrial genome of Didymium and identify six new edited genes as well as one new gene that appears unedited. Next we investigate sequence conservation in the vicinity of editing sites between the two organisms in order to identify sites that harbor the information for the location of editing sites based on increased conservation. Our results imply that the information contained within only nine or ten nucleotides on either side of the editing site (a distance previously suggested through experiments) is not enough to locate the editing sites. Finally, we show that the codon position bias in C insertional RNA editing of these two organisms is correlated with the selection pressure on the respective genes thereby directly testing an evolutionary theory on the origin of this codon bias. Beyond revealing interesting properties of insertional RNA editing in Myxomycetes, our work suggests possible approaches to be used when finding sequence motifs for any biological process fails.
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    ABSTRACT: Amoeboid life forms can be found throughout the evolutionary tree. The greatest proportion of these life forms is found in the Amoebozoa clade, one of the six major eukaryote evolutionary branches. Despite its common origin this clade exhibits a wide diversity of lifestyles including free-living and parasitic species and species with multicellular and multinucleate life stages. In this group, development, cooperation, and social behaviour can be studied in addition to traits common to unicellular organisms. To date, only a few Amoebozoa genomes have been sequenced completely, however a number of expressed sequence tags (ESTs) and complete and draft genomes have become available recently for several species that represent some of the major evolutionary lineages in this clade. This resource allows us to compare and analyse the evolutionary history and fate of branch-specific genes if properly exploited. Despite the large evolutionary time scale since the emergence of the major groups the genomic organization in Amoebozoa has retained common features. The number of Amoebozoa-specific genetic inventions seems to be rather small. The emergence of subgroups is accompanied by gene and domain losses and acquisitions of bacterial gene material. The sophisticated developmental cycles of Myxogastria and Dictyosteliida likely have a common origin and are deeply rooted in amoebozoan evolution. In this review we describe initial approaches to comparative genomics in Amoebozoa, summarize recent findings, and identify goals for further studies.
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