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Extensive transcript diversity and novel upstream open reading frame regulation in yeast.

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.

SUBMITTER: Waern K

PROVIDER: S-EPMC3564994 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Translation of an mRNA in eukaryotes starts at AUG in most cases. Near-cognate codons (NCCs) such as UUG, ACG and AUU are also used as start sites at low levels in S. cerevisiae. Initiation from NCCs or AUGs in the 5’-untranslated regions (UTRs) of mRNAs can lead to translation of upstream open reading frames (uORFs) that might regulate expression of the main ORF (mORF). Although there is some circumstantial evidence that the translation of uORFs can be affected by environmental conditions, little is known about how it is affected by changes in growth temperature. Using reporter assays, we found that changes in growth temperature can affect translation from NCC start sites in yeast cells, suggesting the possibility that gene expression could be regulated by temperature by altering use of different uORF start codons. Using ribosome profiling, we provide evidence that growth temperature regulates the efficiency of translation of nearly 200 uORFs in S. cerevisiae. Of these uORFs, most that start with an AUG codon have increased translational efficiency at 37 ˚C relative to 30 ˚C and decreased efficiency at 20 ˚C. For translationally regulated uORFs starting with NCCs, we did not observe a general trend for the direction of regulation as a function of temperature, suggesting mRNA-specific features can determine the mode of temperature-dependent regulation. Consistent with this conclusion, the position of the uORFs in the 5’-leader relative to the 5’-cap and the start codon of the main ORF correlates with the direction of temperature-dependent regulation of uORF translation. We have identified several novel cases in which changes in uORF translation are inversely correlated with changes in the translational efficiency of the downstream main ORF. Our data suggest that translation of these mRNAs is subject to temperature-dependent, uORF-mediated regulation. Overall, our data suggest that alterations in the translation of specific uORFs by temperature can regulate gene expression in S. cerevisiae.

Project description:BACKGROUND:Translation of an mRNA in eukaryotes starts at an AUG codon in most cases, but near-cognate codons (NCCs) such as UUG, ACG, and AUU can also be used as start sites at low levels in Saccharomyces cerevisiae. Initiation from NCCs or AUGs in the 5'-untranslated regions (UTRs) of mRNAs can lead to translation of upstream open reading frames (uORFs) that might regulate expression of the main ORF (mORF). Although there is some circumstantial evidence that the translation of uORFs can be affected by environmental conditions, little is known about how it is affected by changes in growth temperature. RESULTS:Using reporter assays, we found that changes in growth temperature can affect translation from NCC start sites in yeast cells, suggesting the possibility that gene expression could be regulated by temperature by altering use of different uORF start codons. Using ribosome profiling, we provide evidence that growth temperature regulates the efficiency of translation of nearly 200 uORFs in S. cerevisiae. Of these uORFs, most that start with an AUG codon have increased translational efficiency at 37 °C relative to 30 °C and decreased efficiency at 20 °C. For translationally regulated uORFs starting with NCCs, we did not observe a general trend for the direction of regulation as a function of temperature, suggesting mRNA-specific features can determine the mode of temperature-dependent regulation. Consistent with this conclusion, the position of the uORFs in the 5'-leader relative to the 5'-cap and the start codon of the main ORF correlates with the direction of temperature-dependent regulation of uORF translation. We have identified several novel cases in which changes in uORF translation are inversely correlated with changes in the translational efficiency of the downstream main ORF. Our data suggest that translation of these mRNAs is subject to temperature-dependent, uORF-mediated regulation. CONCLUSIONS:Our data suggest that alterations in the translation of specific uORFs by temperature can regulate gene expression in S. cerevisiae.

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Extensive transcript diversity and novel upstream open reading frame regulation in yeast.

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