Wilhelm, B. T. et al. Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution. Nature 453, 1239-1243

Cancer Research UK Fission Yeast Functional Genomics Group, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK.
Nature (Impact Factor: 41.46). 06/2008; 453(7199):1239-43. DOI: 10.1038/nature07002
Source: PubMed


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.

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    • "Previously characterised quiescence states show a profound alteration in gene expression patterns (Marguerat et al., 2012; Shimanuki et al., 2007; Wilhelm et al., 2008). We identified a large number (>3000) of differentially regulated genes by mRNA-Seq (mRNA isolation followed by next-generation sequencing) in wild-type cells at 4 and 10 h after switch to low-phosphate medium (Fig. S1A, at later time points there was outgrowth of "
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    ABSTRACT: Endocytosis is essential for uptake of many substances into the cell, but how it links to nutritional signalling is poorly understood. Here we show a novel role for endocytosis in regulating the response to low phosphate in Schizosaccharomyces pombe. Loss of function of Myo1, Sla2/End4 or Arp2, proteins involved in the early steps of endocytosis, led to increased proliferation in low phosphate media compared to controls. We show that once cells are deprived of phosphate they undergo a quiescence response that is dependent on the endocytic function of Myo1. Transcriptomic analysis revealed a wide perturbation of gene expression with induction of stress-regulated genes upon phosphate starvation in wildtype but not Δmyo1 cells. Thus, endocytosis plays a pivotal role in mediating the cellular response to nutrients, bridging the external environment and internal molecular functions of the cell.
    Journal of Cell Science 09/2015; 128(20). DOI:10.1242/jcs.171314 · 5.43 Impact Factor
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    • "Based on advances made in the single-cell qPCR field, whole-transcriptome analysis was performed, using microarrays (Kamme et al., 2003; Kurimoto et al., 2006, 2007; Subkhankulova et al., 2008). Subsequently , RNA sequencing (Cloonan et al., 2008; Marioni et al., 2008; Mortazavi et al., 2008; Nagalakshmi et al., 2008; Wilhelm et al., 2008) was quickly adapted for analyzing single cells. The first transcriptomes generated via single-cell RNA-sequencing (scRNA-seq) were published in 2009 (Tang et al., 2009), only 2 years after the first applications of RNA-seq to bulk populations of cells. "
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    ABSTRACT: The differences between individual cells can have profound functional consequences, in both unicellular and multicellular organisms. Recently developed single-cell mRNA-sequencing methods enable unbiased, high-throughput, and high-resolution transcriptomic analysis of individual cells. This provides an additional dimension to transcriptomic information relative to traditional methods that profile bulk populations of cells. Already, single-cell RNA-sequencing methods have revealed new biology in terms of the composition of tissues, the dynamics of transcription, and the regulatory relationships between genes. Rapid technological developments at the level of cell capture, phenotyping, molecular biology, and bioinformatics promise an exciting future with numerous biological and medical applications. Copyright © 2015 Elsevier Inc. All rights reserved.
    Molecular cell 05/2015; 58(4):610-620. DOI:10.1016/j.molcel.2015.04.005 · 14.02 Impact Factor
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    • "Although genome-wide transcriptome sequencing has revealed that ~90% of eukaryotic genomes are transcribed (Wilhelm et al., 2008), only 1–2% of the genome encodes proteins (Birney et al., 2007), suggesting the presence of a large proportion of non-coding RNAs (ncRNAs). The ncR- NAs are classified as housekeeping ncRNAs and regulatory ncRNAs (Kim and Sung, 2012). "
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    ABSTRACT: Recently, long non-coding RNAs (lncRNAs) have been shown to play critical regulatory roles in model plants, such as Arabidopsis, rice, and maize. However, the presence of lncRNAs and how they function in fleshy fruit ripening are still largely unknown because fleshy fruit ripening is not present in the above model plants. Tomato is the model system for fruit ripening studies due to its dramatic ripening process. To investigate further the role of lncRNAs in fruit ripening, it is necessary and urgent to discover and identify novel lncRNAs and understand the function of lncRNAs in tomato fruit ripening. Here it is reported that 3679 lncRNAs were discovered from wild-type tomato and ripening mutant fruit. The lncRNAs are transcribed from all tomato chromosomes, 85.1% of which came from intergenic regions. Tomato lncRNAs are shorter and have fewer exons than protein-coding genes, a situation reminiscent of lncRNAs from other model plants. It was also observed that 490 lncRNAs were significantly up-regulated in ripening mutant fruits, and 187 lncRNAs were down-regulated, indicating that lncRNAs could be involved in the regulation of fruit ripening. In line with this, silencing of two novel tomato intergenic lncRNAs, lncRNA1459 and lncRNA1840, resulted in an obvious delay of ripening of wild-type fruit. Overall, the results indicated that lncRNAs might be essential regulators of tomato fruit ripening, which sheds new light on the regulation of fruit ripening.
    Journal of Experimental Botany 05/2015; 66(15). DOI:10.1093/jxb/erv203 · 5.53 Impact Factor
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