Extensive transcriptional heterogeneity revealed by isoform profiling

1] Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany [2].
Nature (Impact Factor: 41.46). 04/2013; 497(7447). DOI: 10.1038/nature12121
Source: PubMed


Transcript function is determined by sequence elements arranged on an individual RNA molecule. Variation in transcripts can affect messenger RNA stability, localization and translation, or produce truncated proteins that differ in localization or function. Given the existence of overlapping, variable transcript isoforms, determining the functional impact of the transcriptome requires identification of full-length transcripts, rather than just the genomic regions that are transcribed. Here, by jointly determining both transcript ends for millions of RNA molecules, we reveal an extensive layer of isoform diversity previously hidden among overlapping RNA molecules. Variation in transcript boundaries seems to be the rule rather than the exception, even within a single population of yeast cells. Over 26 major transcript isoforms per protein-coding gene were expressed in yeast. Hundreds of short coding RNAs and truncated versions of proteins are concomitantly encoded by alternative transcript isoforms, increasing protein diversity. In addition, approximately 70% of genes express alternative isoforms that vary in post-transcriptional regulatory elements, and tandem genes frequently produce overlapping or even bicistronic transcripts. This extensive transcript diversity is generated by a relatively simple eukaryotic genome with limited splicing, and within a genetically homogeneous population of cells. Our findings have implications for genome compaction, evolution and phenotypic diversity between single cells. These data also indicate that isoform diversity as well as RNA abundance should be considered when assessing the functional repertoire of genomes.

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Available from: Vicent Pelechano, Apr 17, 2014
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    • "(E) NET-seq reads on sense or antisense strands genome-wide in GLU (red) or GAL (blue) for ORF-Ts with peak expression in phases of the YMC indicated. (G) Strand-specific TIF-seq (Pelechano et al., 2013), microarray and NET-seq data at the HMS2:BAT2 locus. Profiles from cells cultured in GLU or GAL on the Watson strand (top) or Crick strand (bottom) are shown. "
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    ABSTRACT: In yeast, many tandemly arranged genes show peak expression in different phases of the metabolic cycle (YMC) or in different carbon sources, indicative of regulation by a bi-modal switch, but it is not clear how these switches are controlled. Using native elongating transcript analysis (NET-seq), we show that transcription itself is a component of bi-modal switches, facilitating reciprocal expression in gene clusters. HMS2, encoding a growth-regulated transcription factor, switches between sense- or antisense-dominant states that also coordinate up- and down-regulation of transcription at neighbouring genes. Engineering HMS2 reveals alternative mono-, di- or tri-cistronic and antisense transcription units (TUs), using different promoter and terminator combinations, that underlie state-switching. Promoters or terminators are excluded from functional TUs by read-through transcriptional interference, while antisense TUs insulate downstream genes from interference. We propose that the balance of transcriptional insulation and interference at gene clusters facilitates gene expression switches during intracellular and extracellular environmental change. DOI: http://dx.doi.org/10.7554/eLife.03635.001
    eLife Sciences 11/2014; 3. DOI:10.7554/eLife.03635 · 9.32 Impact Factor
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    • "To overcome this limitation, tag-based methods able to detect the co-occurrence of a specific transcription start site and a polyadenylation site has been developed. Methods able to determine both ends are called RNA-PET [88] and TIF-Seq [89]. RNA-PET is a paired-end tag approach, where detection of both 3′ and 5′ ends occurs through paired-end sequencing. "
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    ABSTRACT: Technological advances in the sequencing field support in-depth characterization of the transcriptome. Here, we review genome-wide RNA sequencing methods used to investigate specific aspects of gene expression and its regulation, from transcription to RNA processing and translation. We discuss tag-based methods for studying transcription, alternative initiation and polyadenylation events, shotgun methods for detection of alternative splicing, full-length RNA sequencing for the determination of complete transcript structures, and targeted methods for studying the process of transcription and translation. With the ensemble of technologies available, it is now possible to obtain a comprehensive view on transcriptome complexity and the regulation of transcript diversity.
    Cellular and Molecular Life Sciences CMLS 05/2014; 71(18). DOI:10.1007/s00018-014-1637-9 · 5.81 Impact Factor
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    • "Several of these RBPs, including PUF3, PUF4 (Ulbricht & Olivas, 2008) and VTS1 (Rendl et al, 2008), have been identified as regulators of mRNA stability. In addition, the variable 3′ UTR region among alternative 3′ isoforms is enriched for the presence of RBP motifs, highlighting their potential to modulate post‐transcriptional fates of isoforms (Pelechano et al, 2013). To analyze the potential role of RBPs in isoform‐specific RNA stability, we used a published set of RBP motifs (Riordan et al, 2011) and mapped the potential‐binding sites of seven RBPs to the yeast genome. "
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    ABSTRACT: Recent research has uncovered extensive variability in the boundaries of transcript isoforms, yet the functional consequences of this variation remain largely unexplored. Here, we systematically discriminate between the molecular phenotypes of overlapping coding and non-coding transcriptional events from each genic locus using a novel genome-wide, nucleotide-resolution technique to quantify the half-lives of 3' transcript isoforms in yeast. Our results reveal widespread differences in stability among isoforms for hundreds of genes in a single condition, and that variation of even a single nucleotide in the 3' untranslated region (UTR) can affect transcript stability. While previous instances of negative associations between 3' UTR length and transcript stability have been reported, here, we find that shorter isoforms are not necessarily more stable. We demonstrate the role of RNA-protein interactions in conditioning isoform-specific stability, showing that PUF3 binds and destabilizes specific polyadenylation isoforms. Our findings indicate that although the functional elements of a gene are encoded in DNA sequence, the selective incorporation of these elements into RNA through transcript boundary variation allows a single gene to have diverse functional consequences.
    Molecular Systems Biology 02/2014; 10(2):719. DOI:10.1002/msb.135068 · 10.87 Impact Factor
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