Recent data from several organisms indicate that the transcribed portions of genomes are larger and more complex than expected, and that many functional properties of transcripts are based not on coding sequences but on regulatory sequences in untranslated regions or non-coding RNAs1,2,3,4,5,6,7,8,9. Alternative start and polyadenylation sites and regulation of intron splicing add additional dimensions to the rich transcriptional output10,11. This transcriptional complexity has been sampled mainly using hybridization-based methods under one or few experimental conditions. Here we applied direct high-throughput sequencing of complementary DNAs (RNA-Seq), supplemented with data from high-density tiling arrays, to globally sample transcripts of the fission yeast Schizosaccharomyces pombe, independently from available gene annotations. We interrogated transcriptomes under multiple conditions, including rapid proliferation, meiotic differentiation and environmental stress, as well as in RNA processing mutants to reveal the dynamic plasticity of the transcriptional landscape as a function of environmental, developmental and genetic factors. High-throughput sequencing proved to be a powerful and quantitative method to sample transcriptomes deeply at maximal resolution. In contrast to hybridization, sequencing showed little, if any, background noise and was sensitive enough to detect widespread transcription in >90% of the genome, including traces of RNAs that were not robustly transcribed or rapidly degraded. The combined sequencing and strand-specific array data provide rich condition-specific information on novel, mostly non-coding transcripts, untranslated regions and gene structures, thus improving the existing genome annotation. Sequence reads spanning exon–exon or exon–intron junctions give unique insight into a surprising variability in splicing efficiency across introns, genes and conditions. Splicing efficiency was largely coordinated with transcript levels, and increased transcription led to increased splicing in test genes. Hundreds of introns showed such regulated splicing during cellular proliferation or differentiation.
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Raw data are available from ArrayExpress (http://www.ebi.ac.uk/arrayexpress) under accession numbers E-MTAB-5 (sequence data) and E-MTAB-18 (array data). Transcript data-plots are available from our TranscriptomeViewer at http://www.sanger.ac.uk/PostGenomics/S_pombe/.
We thank K. Gould and M. Yamamoto for strains, W. Huber and R. Durbin for advice, and J. Mata, W. Huber, V. Pancaldi, D. Stemple, J.-R. Landry and D. Lackner for comments on the manuscript. B.T.W. was supported by Sanger Postdoctoral and Canadian NSERC fellowships, and S.M. by a fellowship for Advanced Researchers from the Swiss National Science Foundation. This research was funded by Cancer Research UK grant number C9546/A6517 by the Wellcome Trust, and by DIAMONDS, an EC FP6 Lifescihealth STREP (LSHB-CT-2004-512143).
Author Contributions B.T.W., S.M. and J.B. designed and supervised the research and discussed the results; S.W. performed most experiments with help of B.T.W. and S.M.; B.T.W. and S.M. analysed the data with help of F.S., V.W., C.J.P. and J.B.; I.G. and J.R. helped with sequencing; and J.B. drafted the manuscript.
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About this article
Comprehensive profiling of the fission yeast transcription start site activity during stress and media response
Nucleic Acids Research (2019)