Mechanisms of Intron Loss and Gain in the Fission Yeast <i>Schizosaccharomyces</i>

<div><p>The fission yeast, <i>Schizosaccharomyces pombe</i>, is an important model species with a low intron density. Previous studies showed extensive intron losses during its evolution. To test the models of intron loss and gain in fission yeasts, we conducted a comparative genomic analysis in four <i>Schizosaccharomyces</i> species. Both intronization and de-intronization were observed, although both were at a low frequency. A de-intronization event was caused by a degenerative mutation in the branch site. Four cases of imprecise intron losses were identified, indicating that genomic deletion is not a negligible mechanism of intron loss. Most intron losses were precise deletions of introns, and were significantly biased to the 3′ sides of genes. Adjacent introns tended to be lost simultaneously. These observations indicated that the main force shaping the exon-intron structures of fission yeasts was precise intron losses mediated by reverse transcriptase. We found two cases of intron gains caused by tandem genomic duplication, but failed to identify the mechanisms for the majority of the intron gain events observed. In addition, we found that intron-lost and intron-gained genes had certain similar features, such as similar Gene Ontology categories and expression levels.</p></div

Phylogenetic relationship of the fission yeasts and six outgroup fungal species.

<p>The tree was constructed from a study on comparative genomics of fission yeasts <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061683#pone.0061683-Rhind1" target="_blank">[47]</a> and the NCBI Taxonomy database (<a href="http://www.ncbi.nlm.nih.gov/taxonomy" target="_blank">http://www.ncbi.nlm.nih.gov/taxonomy</a>). It is not scaled according to phylogenetic distances.</p

The number and rate of intron loss and gain in fission yeasts.

a<p>Species name abbreviations: <i>S. cryophilus</i> (<i>Scry</i>), <i>S. octosporus</i> (<i>Soct</i>), <i>S. pombe</i> (<i>Spom</i>).</p>b<p>Only introns that were not filtered out by our criteria were counted, including conserved introns and unique introns. Therefore, they are smaller than the total numbers of annotated introns.</p>c<p>The divergence time between each species and the nodes were obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061683#pone.0061683-Rhind1" target="_blank">[47]</a>. Data are shown in millions of years.</p>d<p>Introns lost or gained at the ancestor nodes were counted only once.</p>e<p>The rates of intron loss are shown as number of lost introns per year per intron.</p

Adjacent introns tend to be lost together in fission yeasts.

<p>The probability distribution of all possible numbers of adjacent lost intron pairs is shown, with the observed pattern marked by a circle. The probabilities exceeding the observed numbers of lost intron pairs were small and, therefore, adjacent introns tend to be lost together more frequently than by chance. Lost introns are categorized by A) <i>S. pombe</i>, B) <i>S. japonicus</i>, C) Ancestor of <i>S. cryophilus</i>, <i>S. octosporus</i> and <i>S. pombe</i>, D) Ancestor of <i>S. cryophilus</i> and <i>S. octosporus</i>.</p

Comparison of the relative positions between lost introns and conserved introns^a.

a<p>The relative position was defined as intron position divided by the length of the coding sequence from the 5′ end. For introns lost at the ancestor nodes, the relative position was calculated in each of the species. Species name abbreviations: <i>S. cryophilus</i> (<i>Scry</i>), <i>S. octosporus</i> (<i>Soct</i>), <i>S. pombe</i> (<i>Spom</i>).</p>b<p>All the relative intron positions were grouped by lost introns and conserved introns and a Mann-Whitney <i>U</i> test was used to calculate the <i>P</i> values.</p>c<p>For each intron-lost gene, the median positions of lost introns and conserved introns were paired and a Wilcoxon signed rank test was used to calculate the <i>P</i> values. Intron-lost genes with no conserved introns were not counted; therefore, the sample sizes were smaller than grouped comparisons.</p

Detection of intronization and de-intronization.

<p>Both intronization and de-intronization are characterized by introns neighboring large gaps while the surrounding coding regions remain well aligned. These two can be distinguished by the presence or absence of the introns in other outgroup species (A). The conserved surrounding coding regions are marked in yellow and the exonized or intronized regions are marked in red. Introns are represented as stars. The protein alignments show a case of intronization in <i>S. pombe</i> (B) and a case of de-intronization in <i>S. cryophilus</i> (C). Intron phases are marked as 0, 1, 2 or ∼ (absence of an intron). Species names abbreviations: <i>S. cryophilus</i> (<i>Scry</i>), <i>S. octosporus</i> (<i>Soct</i>), <i>S. pombe</i> (<i>Spom</i>), and <i>S. japonicus</i> (<i>Sjap</i>).</p

Cases of imprecise intron deletion in fission yeasts.

<p>The alignments of DNA sequences around imprecise intron deletion regions are shown. Exon sequences are shown in upper case while intron sequences are shown in lower case. Exonic sequence indels accompanying intron loss are marked in red. Internal regions in long intron sequences are marked by “//”. Species name abbreviations: <i>S. cryophilus</i> (<i>Scry</i>), <i>S. octosporus</i> (<i>Soct</i>), <i>S. pombe</i> (<i>Spom</i>), and <i>S. japonicus</i> (<i>Sjap</i>).</p

Intronization and de-intronization events in fission yeasts.

<p>A) Intronization occurred in the <i>SPBC29A10.02</i> gene of <i>S. pombe</i>. The intronized region is marked by underlining and variations in splice sites are marked in gray. Alignment of gene <i>SPBC29A10.02</i> with its related EST is shown below. B) De-intronization occurred in the <i>SPOG_00055</i> gene of <i>S. cryophilus</i>. Alignment of <i>SPOG_00055</i> with its orthologs shows a de-intronization event, with the exonized region marked by underlining. Alignment of gene <i>SPOG_00055</i> with its related EST is shown below. C) The consensus sequence (YTRAY) of branch sites in fission yeasts. Branch site sequences were detected using ICAT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061683#pone.0061683-Drabenstot1" target="_blank">[53]</a> and consensus sequences were generated using Weblogo <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061683#pone.0061683-Crooks1" target="_blank">[54]</a>. D) The degraded branch sites of <i>SPOG_00055</i> compared with its orthologous intron regions. The branch sites predicted by ICAT are marked by underlining and the consensus regions in the branch sites are in bold. Mutations are marked in gray. The introns are shown in lower case while exonic sequences are presented in upper case. Species name abbreviations: <i>S. cryophilus</i> (<i>Scry</i>), <i>S. octosporus</i> (<i>Soct</i>), <i>S. pombe</i> (<i>Spom</i>), and <i>S. japonicus</i> (<i>Sjap</i>).</p

Overrepresented GO categories in intron-lost and intron-gained genes.

a<p>A hypergeometric test was used and only GO categories with <i>P</i> values lower than 0.05 were shown.</p>*<p>GO categories that were both present in intron-lost and intron-gained genes. The null hypothesis is that the intron-lost and intron-gained genes were randomly clustered among the GO categories. However, three out of four GO categories of intron-gained genes were shared by intron-lost genes, which implied that intron loss and gain in fission yeast might share some similar mechanisms.</p

Number of putative intronization and de-intronization events in fission yeasts.

a<p>Unable to distinguish between intronization and de-intronization because of a lack of outgroup evidence.</p>b<p>For intronization, “Supported by transcriptome data” means that the novel intron region is spliced in at least one transcript. “Disproved by transcriptome data” means that the novel intron region is retained in all the related transcripts. For de-intronization, “Supported by transcriptome data” means the novel exon region is retained in at least one transcript. “Disproved by transcriptome data” means the novel exon region is spliced in all transcripts and is thus an unannotated intron. If no transcripts cover the novel intron or exon region, it is defined as “Lacking related transcript data”.</p