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Extensive Transcript Diversity and Novel Upstream Open Reading Frame Regulation in Yeast

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520.
G3-Genes Genomes Genetics (Impact Factor: 2.51). 02/2013; 3(2):343-52. DOI: 10.1534/g3.112.003640
Source: PubMed

ABSTRACT To understand the diversity of transcripts in yeast (Saccharomyces cerevisiae) we analyzed the transcriptional landscapes for cells grown under 18 different environmental conditions. Each sample was analyzed using RNA-sequencing, and a total of 670,446,084 uniquely mapped reads and 377,263 poly-adenylated end tags were produced. Consistent with previous studies, we find that the majority of yeast genes are expressed under one or more different conditions. By directly comparing the 5' and 3' ends of the transcribed regions, we find extensive differences in transcript ends across many conditions, especially those of stationary phase, growth in grape juice, and salt stimulation, suggesting differential choice of transcription start and stop sites is pervasive in yeast. Relative to the exponential growth condition (i.e., YPAD), transcripts differing at the 5' ends and 3' ends are predicted to differ in their annotated start codon in 21 genes and their annotated stop codon in 63 genes. Many (431) upstream open reading frames (uORFs) are found in alternate 5' ends and are significantly enriched in transcripts produced during the salt response. Mutational analysis of five genes with uORFs revealed that two sets of uORFs increase the expression of a reporter construct, indicating a role in activation which had not been reported previously, whereas two other uORFs decreased expression. In addition, RNA binding protein motifs are statistically enriched for alternate ends under many conditions. Overall, these results demonstrate enormous diversity of transcript ends, and that this heterogeneity is regulated under different environmental conditions. Moreover, transcript end diversity has important biological implications for the regulation of gene expression. In addition, our data also serve as a valuable resource for the scientific community.

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    • "Changes in codon usage have been previously implicated in yeast gene regulatory evolution (Man and Pilpel 2007). More recent studies have highlighted important translation regulatory roles of transcript leaders in S. cerevisiae (Ingolia et al. 2009; Brar et al. 2011; Rojas-Duran and Gilbert 2012; Waern and Snyder 2013). Our results indicate that interspecies differences in transcript leaders appear to play a larger role than codon bias. "
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    • "Changes in codon usage have been previously implicated in yeast gene regulatory evolution (Man and Pilpel 2007). More recent studies have highlighted important translation regulatory roles of transcript leaders in S. cerevisiae (Ingolia et al. 2009; Brar et al. 2011; Rojas-Duran and Gilbert 2012; Waern and Snyder 2013). Our results indicate that interspecies differences in transcript leaders appear to play a larger role than codon bias. "
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    • "In S. cerevisiae, such pervasive transcripts include the cryptic unstable transcripts (CUTs), stable unannotated transcripts (SUTs), Xrn1-sensitive unstable transcripts (XUTs), and meiotic unannotated transcripts (MUTs) (Wyers et al. 2005; Xu et al. 2009; Lardenois et al. 2011; van Dijk et al. 2011). Additionally, alterations in the prevalence of intergenic transcripts and transcript start and end sites have been observed under different growth and stress conditions (Xu et al. 2009; Waern and Snyder 2013). Since the vast majority of these cryptic transcripts and transcript isoforms have unknown functions, it has been speculated that they may represent inherent sloppiness of the transcriptional process, referred to as “transcriptional noise” (Struhl 2007). "
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    ABSTRACT: The major function of eukaryotic RNA polymerase III is to transcribe transfer RNA, 5S ribosomal RNA, and other small non-protein coding RNA molecules. Assembly of the RNA polymerase III complex on chromosomal DNA requires the sequential binding of transcription factor complexes TFIIIC and TFIIIB. Recent evidence has suggested that in addition to producing RNA transcripts, chromatin assembled RNA polymerase III complexes may mediate additional nuclear functions that include chromatin boundary, nucleosome phasing, and general genome organization activities. This study provides evidence of another such "extra-transcriptional" activity of assembled RNA polymerase III complexes, which is the ability to block progression of intergenic RNA polymerase II transcription. We demonstrate that the RNA polymerase III complex bound to the tRNA gene upstream of the Saccharomyces cerevisiae ATG31 gene protects the ATG31 promoter against readthrough transcriptional interference from the upstream non-coding intergenic SUT467 transcription unit. This protection is predominately mediated by binding of the TFIIIB complex. When TFIIIB binding to this tRNA gene is weakened, an extended SUT467-ATG31 readthrough transcript is produced, resulting in compromised ATG31 translation. Since the ATG31 gene product is required for autophagy, strains expressing the readthrough transcript exhibit defective autophagy induction and reduced fitness under autophagy-inducing nitrogen starvation conditions. Given the recent discovery of widespread pervasive transcription in all forms of life, protection of neighboring genes from intergenic transcriptional interference may be a key extra-transcriptional function of assembled RNA polymerase III complexes and possibly other DNA binding proteins.
    Genetics 12/2013; 196(2). DOI:10.1534/genetics.113.160093 · 4.87 Impact Factor
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