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. 2010;11(8):R87.
doi: 10.1186/gb-2010-11-8-r87. Epub 2010 Aug 26.

Strand-specific RNA sequencing reveals extensive regulated long antisense transcripts that are conserved across yeast species

Moran Yassour  1 , Jenna Pfiffner, Joshua Z Levin, Xian Adiconis, Andreas Gnirke, Chad Nusbaum, Dawn-Anne Thompson, Nir Friedman, Aviv Regev
Affiliations

Strand-specific RNA sequencing reveals extensive regulated long antisense transcripts that are conserved across yeast species

Moran Yassour et al. Genome Biol. 2010.

Abstract

Background: Recent studies in budding yeast have shown that antisense transcription occurs at many loci. However, the functional role of antisense transcripts has been demonstrated only in a few cases and it has been suggested that most antisense transcripts may result from promiscuous bi-directional transcription in a dense genome.

Results: Here, we use strand-specific RNA sequencing to study anti-sense transcription in Saccharomyces cerevisiae. We detect 1,103 putative antisense transcripts expressed in mid-log phase growth, ranging from 39 short transcripts covering only the 3' UTR of sense genes to 145 long transcripts covering the entire sense open reading frame. Many of these antisense transcripts overlap sense genes that are repressed in mid-log phase and are important in stationary phase, stress response, or meiosis. We validate the differential regulation of 67 antisense transcripts and their sense targets in relevant conditions, including nutrient limitation and environmental stresses. Moreover, we show that several antisense transcripts and, in some cases, their differential expression have been conserved across five species of yeast spanning 150 million years of evolution. Divergence in the regulation of antisense transcripts to two respiratory genes coincides with the evolution of respiro-fermentation.

Conclusions: Our work provides support for a global and conserved role for antisense transcription in yeast gene regulation.

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Figures

Figure 1
Figure 1
Strand-specific RNA-seq identifies 1,103 antisense units associated with stationary phase, stress, and meiosis genes in S. cerevisiae. (a) Typical short antisense (Unit3689, antisense to NOP10). Shown are reads mapped from a standard cDNA sequencing library [15] (yellow), and from the strand-specific library prepared and run side-by-side on the same flow cell (green: forward reads above, reverse reads below). All coverage tracks were normalized to the total number of reads mapped, and are shown up to a threshold of 3 × 10-8 of total mapped reads (genome-wide). Units were called from the strand-specific library (blue units, known genes; orange, putative antisense), and are shown along with the manually curated units (red) and the known gene annotations from the SGD (gray). (b) Typical long antisense (ManualUnit225, antisense to MBR1). Tracks are as in (a). The figures are shown using the Integrative Genome Viewer [36].
Figure 2
Figure 2
Quantitative expression measurements of putative antisense units and the corresponding sense genes in S. cerevisiae. (a) Strand-specific qRT-PCR measurements of six pairs of known sense genes and their putative antisense units in comparing mid-log and early stationary phase (the y-axis shows the log2 ratio of expression in early stationary phase versus mid-log). Error bars indicate the standard deviation between biological replicates and different primers. (b) nCounter [20] measurements of nine representative putative antisense units, comparing mid-log to early stationary phase, stationary phase, heat shock and salt stress (the y-axis is as in (a) for the examined condition). Error bars indicate the standard deviation between biological replicates. (c) nCounter measurement for 67 tested sense-antisense pairs in early stationary phase (left) and heat shock (right), each relative to a mid-log (no stress) control. The columns marked 'S' and 'A' represent the sense and antisense change, respectively. Red, induced; green, repressed; black, no change. The names of genes highlighted in the main text are shown in red.
Figure 3
Figure 3
Effect of Rrp6 and Hda2 on antisense transcript levels and sense-antisense regulation. (a,b) The distribution of changes in expression levels (x-axis) for sense (blue) and antisense (orange) transcripts in the Δrrp6 (a) and Δhda2 (b) mutants compared to the wild type (wt). In the Δrrp6 mutant (a) there is a mild increase in antisense levels and decrease in sense levels. No such changes are observed in the Δhda2 mutant (b). (c) Negative correlation between change in antisense transcript (y-axis) and in sense transcript (x-axis) in the Δrrp6 mutant relative to the wild-type strain. (d) Similarity in differential sense gene expression from mid-log to early stationary phase between the wild type (x-axis) and the Δrrp6 mutant (y-axis).
Figure 4
Figure 4
Conservation of the presence and regulation of antisense units in Hemiascomycota. Shown are the differential expression values of antisense and sense units comparing mid-log and early stationary phase across S. cerevisiae and the five other species (red, higher in early stationary phase; green, lower in early stationary phase; black, no change; hatched, no candidate orthologous contig; grey, no antisense transcription detected in species). A phylogenetic tree of the species included in this study [27] is shown above (the star indicates the WGD).
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