Placozoan <i>cox1</i> “mRNA editing” scenario.

<p>The shown scenario is based on <i>Trichoplax adhaerens</i> EST data (Burger <i>et al</i>., 2009). The figure only shows <i>cox1</i> exons 6 and 7 and the intron between them (following NC_008151). After splicing of exons the “U” at the 3’ end of exon 6 is converted to a “C” by mRNA editing. Exons and introns are illustrated in yellow and blue, respectively. mRNA editing (“U-to-C”) is illustrated by red lightning. For further explanations see text and Burger <i>et al</i>., 2009.</p

Placozoan <i>cox1</i> “micro exon” scenario.

<p>The scenario is based on Placozoa sp. H2 “Panama” RNA-seq data. Exon/intron color codes are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177959#pone.0177959.g002" target="_blank">Fig 2</a>. Exon 6¹ represents a truncated exon 6 (following Burger <i>et al</i>., 2009), which is indicated by the superscript 1. Subsequent exons/introns also differ in boundaries and/or numbering from the annotation by Burger <i>et al</i>., 2009 (likewise indicated by a superscript 1). The former intron 6 is now split into two introns (intron 6¹ and 7¹, respectively) flanking the newly identified micro exon 7¹, which has been identified in this study. Splicing of exon 6¹, micro exon 7¹ and exon 8¹ (formerly exon 7, Burger <i>et al</i>., 2009) leads to an in-frame coding sequence (CDS) with the intact CAT triplet coding for the functionally indispensable histidine at the respective position.</p

Deep RNA sequencing reveals the smallest known mitochondrial micro exon in animals: The placozoan <i>cox1</i> single base pair exon

<div><p>The phylum Placozoa holds a key position for our understanding of the evolution of mitochondrial genomes in Metazoa. Placozoans possess large mitochondrial genomes which harbor several remarkable characteristics such as a fragmented <i>cox1</i> gene and trans-splicing <i>cox1</i> introns. A previous study also suggested the existence of <i>cox1</i> mRNA editing in <i>Trichoplax adhaerens</i>, yet the only formally described species in the phylum Placozoa. We have analyzed RNA-seq data of the undescribed sister species, Placozoa sp. H2 (“Panama” clone), with special focus on the mitochondrial mRNA. While we did not find support for a previously postulated <i>cox1</i> mRNA editing mechanism, we surprisingly found two independent transcripts representing intermediate <i>cox1</i> mRNA splicing stages. Both transcripts consist of partial <i>cox1</i> exon as well as overlapping intron fragments. The data suggest that the <i>cox1</i> gene harbors a single base pair (cytosine) micro exon. Furthermore, conserved group I intron structures flank this unique micro exon also in other placozoans. We discuss the evolutionary origin of this micro exon in the context of a self-splicing intron gain in the <i>cox1</i> gene of the last common ancestor of extant placozoans.</p></div

Conserved splicing sites and intron motifs in the placozoan <i>cox1</i> gene.

<p>A) Partial alignment of the placozoan <i>cox1</i> intron containing the predicted <i>cox1</i> micro exon. Although the overall pairwise sequence similarity of respective introns is low, the micro exon splicing motif can be found in all known placozoan mitochondrial genomes (for accession numbers see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177959#sec003" target="_blank">Material and methods</a>). Numbers represent nucleotide position in the intron alignment. B) RNAweasel intron predictions annotated on partial placozoan <i>cox1</i> sequences. Conserved core group IB intron structures predicted by RNAweasel are illustrated in orange. In all known placozoan species the <i>cox1</i> micro exon is flanked by two independent introns, respectively.</p

Schematic <i>cox1</i> transcript mapping.

<p>Shown are transcripts W, X and Y (assembled from multiple RNA-seq reads) and transcript Z (hypothetical transcript) mapped onto the partial <i>cox1</i> region of Placozoa sp. H2 “Panama”. Exon/intron color codes as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177959#pone.0177959.g001" target="_blank">Fig 1</a>. The superscript 1 indicates differences in the <i>cox1</i> annotation compared to Burger <i>et al</i>., 2009 (see text). Continuous reads/transcripts are indicated by yellow and black connector lines, respectively. The upper transcripts (W) and (X) represent intermediate splicing stages and transcripts from both directions overlap at the micro exon “C” (marked in yellow). Transcript (Y) represents the <i>cox1</i> mature mRNA sequence (in agreement with EST data from <i>Trichoplax adhaerens</i>). Transcript (Z) represents a hypothetical pre-mRNA-editing transcript (following Burger <i>et al</i>., 2009) which has not been found in our RNA-seq data. The putative mRNA editing site in transcript (Z) is indicated by a red “T”. The <i>Trichoplax adhaerens</i> cDNA read supporting the micro exon as well as the intron 7¹ splice sites is illustrated at the bottom.</p

Comparison of placozoan <i>cox1</i> annotations.

<p>A) Positions and lengths of <i>cox1</i> exons in <i>Trichoplax adhaerens</i> according to Burger <i>et al</i>. 2009 (NC_008151.2). B) Differences in numbering, position and length of <i>cox1</i> exons in <i>Trichoplax adhaerens</i> considering the micro exon. C) Different positions of the <i>cox1</i> micro exon in all known placozoan mitochondrial genomes.</p

Mapping of RNA-seq reads on the partial Placozoa sp. H2 "Panama" <i>cox1</i> gene locus containing the micro exon.

<p>The <i>cox1</i> structure is given in the upper part. Exon/intron color codes are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177959#pone.0177959.g002" target="_blank">Fig 2</a>. Mapping regions I, II and III are indicated by dotted lines and are enlarged below. Reads corresponding to transcript W (comprising exon 6¹, micro exon 7¹ and intron 7¹) span region I and II while reads corresponding to transcript X (comprising intron 6¹, micro exon 7¹ and exon 8¹) span region II and III, respectively. Continuous RNA-seq reads are connected by dashed lines (consequence of the applied gapped mapping procedure).</p

The evolutionary origin of the <i>cox1</i> micro exon in Placozoa.

<p>Exon/intron color codes are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177959#pone.0177959.g002" target="_blank">Fig 2</a>. In the ancestral state the continuous CAT triplet was located in the hypothetical exon 7⁰ (comprising the later micro exon 7¹ and exon 8¹, this study). During course of evolution, the “C” of the conserved CAT triplet was isolated from the hypothetical exon 7⁰ by the insertion of a self-splicing intron 7¹.</p

Locations of genotyped placozoans.

<p>Locations of genotyped placozoans.</p

Diagnostic morphological characters identified in the Placozoa.

<p>Diagnostic morphological characters identified in the Placozoa.</p