Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution.
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 RNAs. Alternative start and polyadenylation sites and regulation of intron splicing add additional dimensions to the rich transcriptional output. 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.
Wilhelm
Brian T
BT
Cancer Research UK Fission Yeast Functional Genomics Group, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK.
Marguerat
Samuel
S
Watt
Stephen
S
Schubert
Falk
F
Wood
Valerie
V
Goodhead
Ian
I
Penkett
Christopher J
CJ
Rogers
Jane
J
Bähler
Jürg
J
D017398:Alternative Splicing / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D005057:Eukaryotic Cells / Q000378:metabolism*; D005091:Exons / Q000235:genetics; D020869:Gene Expression Profiling; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal / Q000235:genetics; D007438:Introns / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D012319:RNA Polymerase II / Q000378:metabolism; D012331:RNA, Fungal / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic / Q000235:genetics
D012331:RNA, Fungal; D012333:RNA, Messenger; D029702:Schizosaccharomyces pombe Proteins; D012319:RNA Polymerase II