Dynamic transcriptome of Schizosaccharomyces pombe shown by RNA-DNA hybrid mapping

Abstract

We have determined the high-resolution strand-specific transcriptome of the fission yeast S. pombe under multiple growth conditions using a novel RNA-DNA hybridization mapping (HybMap) technique. HybMap uses an antibody against an RNA-DNA hybrid to detect RNA molecules hybridized to a high-density DNA oligonucleotide tiling microarray. HybMap showed exceptional dynamic range and reproducibility, and allowed us to identify strand-specific coding, noncoding and structural RNAs, as well as previously unknown RNAs conserved in distant yeast species. Notably, we found that virtually the entire euchromatic genome (including intergenics) is transcribed, with heterochromatin dampening intergenic transcription. We identified features including large numbers of condition-specific noncoding RNAs, extensive antisense transcription, new properties of antisense transcripts and induced divergent transcription. Furthermore, our HybMap data informed the efficiency and locations of RNA splicing genome-wide. Finally, we observed strand-specific transcription islands around tRNAs at heterochromatin boundaries inside centromeres. Here, we discuss these new features in terms of organism fitness and transcriptome evolution.

Figure 1

Extensive transcription of the S. pombe genome. (a) S. pombe probes were categorized according to genomic feature and classified into sense and antisense probes on the basis of gene orientation. (b) The percentage of the genome that is transcribed (y axis) at different signal thresholds (x axis), shown for two different units of measurement, genes or base pairs.

Figure 2

Features of transcription and splicing in euchromatin. High-resolution views of different loci are depicted. The physical map is presented in a strand-specific manner, with currently annotated exons (gray boxes) and introns (gray lines). RNA signal intensities for probes along the top/forward (blue) and bottom/reverse (orange) strands are provided (probe centered). The background (dashed line) and the baseline (solid line) are shown for each strand. (a) A locus that generally conforms to current annotation. Brackets denote regions where signal drops below baseline on only one strand, near the TSS. (b) A locus that deviates from current annotation at several locations. Unannotated UTRs and antisense transcription are evident. (c) A typical locus, where signal intensity drops in accordance with annotated introns. (d) snoR54 signal reflects its transcription and processing from an intron within gua1. snoRNAs are highly stable and typically accumulate to levels well beyond their host RNAs. (e) tRNA genes are multicopy and highly transcribed, resulting in a saturated signal. We note that because of the high sequence conservation among tRNAs, their signal cannot be uniquely attributed.

Figure 3

Quantitation of transcription fragments and potential noncoding RNAs. Transfrags are quantified on the basis of threshold criteria. New transfrags identified by HybMap are denoted by hatched boxes. (a) Recently identified ncRNA TER1 is observed as a transfrag in our dataset. (b) Genome-wide analyses yield similar high numbers of transfrags in each condition. (c-e) Data from poly(A)-enriched RNA (overlaid blue line) and a Pol II ChIP (green line) provide evidence for the existence of potential ncRNAs. Transfrags are found in three general genomic environments: in intergenic regions (c), in the antisense strand of known genes (d) and in intronic regions (e).

Figure 4

Transcriptomes and loci derived from alternative growth conditions. Signals from alternative growth conditions (colored line) are overlaid on standard conditions (bars). (a-f) Under heat stress (a,b), minimal medium (c,d) or MMS treatment (e,f); the expression of particular genes (a,c,e) and transfrags (b,d,f) is induced.

Figure 5

Features of antisense transcription. (a) Correlation of sense and antisense transcription. The 432 genes that differ at least fourfold in heat shock from standard conditions are plotted. For each gene, the median change for the sense strand was plotted against the antisense strand. (b,c) Signal in standard conditions (bars) overlaid with poly(A)-enriched data (overlaid blue line). Antisense transcription may be generally divided into two classes comprised of genes with negligible polyadenylated antisense transcripts (the vast majority) (b) and rare genes with polyadenylated antisense transcripts (c).

Figure 6

Poly(A) RNA shows divergent transcription and previously unknown RNA transcripts. (a) We depict the locus in Figure 2b overlaid with poly(A) data (solid purple line). Brackets denote three short transcripts diverging from hsp60, cip2 and SPAC630.07c. Notably, these transcripts would not be distinguishable in total RNA, as a result of the prevalence of nonadenylated antisense. (b,c) A polyadenylated transcript clearly corresponds to each of the two transfrags most conserved among the related yeast species.

Figure 7

Transcriptional features of heterochromatic loci and flanking regions. Signals derived from the top/forward and bottom/reverse strands are overlaid, to enable direct comparison. A colored solid line (underneath) indicates the locations of unique (green) and non-unique (purple) probes. (a) Transcriptional repression near telomeres. The physical map of the subteleomere of chromosome 1 is accompanied by maps of the heterochromatic mark H3K9me2 (ChIP data, black) and a mark correlated with transcribed ORFs, H3K36me3 (ChIP data, green). Notably, as the signal from H3K9me2 increases, intergenic transcription approaches the background level. (b) Transcription within the centromere of chromosome 1. Notably, both strands are transcribed in the regions flanking the dh1 repeats. The opposite strand flanking the highly expressed tRNAs is transcribed. Results for chromosomes 2 and 3 are provided in Supplementary Figure 7.