Identification of putative regulatory upstream ORFs in the yeast genome using heuristics and evolutionary conservation

Cvijović, Marija; Dalevi, Daniel; Bilsland, Elizabeth; Kemp, Graham; Sunnerhagen, Per

doi:10.1186/1471-2105-8-295

Cited by 46 publications

(62 citation statements)

References 40 publications

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“…Assessment of the GCN4 mRNAs in 12 fungal species revealed that uORFs range from three to six in number and are not positionally conserved (25,89). Furthermore, the mechanism of GCN4 translation control for Candida albicans is reliant upon bypass of a single inhibitory uORF whereas GCN4 in S. cerevisiae relies on a mechanism involving delayed translation reinitiation that features multiple uORFs (Fig.…”

Section: Evolutionary Conservation Of Uorf-mediated Translation Mechamentioning

confidence: 99%

Upstream Open Reading Frames Differentially Regulate Gene-specific Translation in the Integrated Stress Response

Young

Wek

2016

Journal of Biological Chemistry

337

313

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Translation regulation largely occurs during initiation, which features ribosome assembly onto mRNAs and selection of the translation start site. Short, upstream ORFs (uORFs) located in the 5-leader of the mRNA can be selected for translation. Multiple transcripts associated with stress amelioration are preferentially translated through uORF-mediated mechanisms during activation of the integrated stress response (ISR) in which phosphorylation of the ␣ subunit of eIF2 results in a coincident global reduction in translation initiation. This review presents key features of uORFs that serve to optimize translational control that is essential for regulation of cell fate in response to environmental stresses.Multiple genome-wide analyses, including those utilizing ribosome and polysome profiling and mass spectrometry approaches, have provided evidence demonstrating that translation is a major regulator of gene expression (1-5). In addition to protein coding sequences (CDSs), 2 another class of ORFs suggested to be translated at high frequency consists of short, upstream ORFs (uORFs) that are located within the 5Ј-leader of mRNAs (3-6). Over 40% of mammalian mRNAs contain uORFs, illustrating that uORFs are prevalent genome-wide and can serve as major regulators of translation (5,7,8). Approximation of uORF prevalence has relied upon the use of an AUG to denote the uORF start codon; however, recent ribosome profiling studies indicate that non-canonical initiation codons (e.g. CUG, UUG, and GUG) can also serve as competent sites of translation initiation (3, 4, 6). These findings suggest that the magnitude of uORF prevalence and the contribution of uORF translation in the regulation of gene expression have likely been underestimated.Typically, uORFs are considered to be inhibitors of downstream translation initiation at CDSs. The inhibitory effect of uORFs is attributed to the fact that in eukaryotes the 43S preinitiation complex binds to the 5Ј-cap structure of the mRNA, then scans processively 5Ј to 3Ј and initiates translation at the first encountered initiation codon that is in an optimal context (9). The 43S preinitiation complex is composed of multiple factors including eIF3, eIF1, eIF1A, the eIF2⅐GTP⅐Met-tRNA i Met ternary complex, and the small 40S ribosomal subunit (10). Disassociation of the eIF2 ternary complex and other critical initiation factors during translation of constitutively repressing uORFs is suggested to be the cause of the low levels of subsequent translation reinitiation at downstream coding sequences.Although uORFs can serve as repressors of CDS translation in a constitutive manner, there are also examples of uORFs that serve as dampeners in a controlled fashion or even promote translation initiation at the CDS in response to environmental stresses (4, 5, 11). Based on these studies, uORFs can have the following core properties that are critical for translational control (Fig. 1). 1) They enhance reinitiation after uORF translation, allowing for degrees of translation initiation at the downstre...

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Section: Evolutionary Conservation Of Uorf-mediated Translation Mechamentioning

confidence: 99%

Upstream Open Reading Frames Differentially Regulate Gene-specific Translation in the Integrated Stress Response

Young

Wek

2016

Journal of Biological Chemistry

337

313

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“…Previous comparative genomics of yeast uORFs assumed that entire intergenic regions were transcribed (Cvijovićet al 2007) or that other species use the same transcription start sites found in S. cerevisiae (Zhang and Dietrich 2005;Selpi et al 2009). By annotating the TLs from four Saccharomyces species, we defined the "search space" for uORFs in each species.…”

Section: Conserved Non-aug Uorfs In Yeastmentioning

confidence: 99%

Conserved non-AUG uORFs revealed by a novel regression analysis of ribosome profiling data

et al. 2017

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Upstream open reading frames (uORFs), located in transcript leaders (5′ UTRs), are potent cis-acting regulators of translation and mRNA turnover. Recent genome-wide ribosome profiling studies suggest that thousands of uORFs initiate with non-AUG start codons. Although intriguing, these non-AUG uORF predictions have been made without statistical control or validation; thus, the importance of these elements remains to be demonstrated. To address this, we took a comparative genomics approach to study AUG and non-AUG uORFs. We mapped transcription leaders in multiple Saccharomyces yeast species and applied a novel machine learning algorithm (uORF-seqr) to ribosome profiling data to identify statistically significant uORFs. We found that AUG and non-AUG uORFs are both frequently found in Saccharomyces yeasts. Although most non-AUG uORFs are found in only one species, hundreds have either conserved sequence or position within Saccharomyces. uORFs initiating with UUG are particularly common and are shared between species at rates similar to that of AUG uORFs. However, non-AUG uORFs are translated less efficiently than AUG-uORFs and are less subject to removal via alternative transcription initiation under normal growth conditions. These results suggest that a subset of non-AUG uORFs may play important roles in regulating gene expression.

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“…uAUGs are thought to negatively affect the efficiency of translation initiation at the downstream ORF, but the generality of this effect in yeast has been a subject of debate, partially because previous analyses were restricted to a handful of uAUGs (Cvijovi c et al 2007;Lawless et al 2009). To assess the generality of uAUGmediated translational repression in rapidly dividing yeast, we examined the polysomal distribution of 773 TL species that contained one or more uAUGs compared with 3143 TL species that lacked uAUGs.…”

Section: Uaugs Are Conserved Inhibitory Elements For Translationmentioning

confidence: 99%

Roles for transcript leaders in translation and mRNA decay revealed by transcript leader sequencing

2013

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Transcript leaders (TLs) can have profound effects on mRNA translation and stability. To map TL boundaries genomewide, we developed TL-sequencing (TL-seq), a technique combining enzymatic capture of m 7 G-capped mRNA 59 ends with high-throughput sequencing. TL-seq identified mRNA start sites for the majority of yeast genes and revealed many examples of intragenic TL heterogeneity. Surprisingly, TL-seq identified transcription initiation sites within 6% of proteincoding regions, and these sites were concentrated near the 59 ends of ORFs. Furthermore, ribosome density analysis showed these truncated mRNAs are translated. Translation-associated TL-seq (TATL-seq), which combines TL-seq with polysome fractionation, enabled annotation of TLs, and simultaneously assayed their function in translation. Using TATLseq to address relationships between TL features and translation of the downstream ORF, we observed that upstream AUGs (uAUGs), and no other upstream codons, were associated with poor translation and nonsense-mediated mRNA decay (NMD). We also identified hundreds of genes with very short TLs, and demonstrated that short TLs were associated with poor translation initiation at the annotated start codon and increased initiation at downstream AUGs. This frequently resulted in out-of-frame translation and subsequent termination at premature termination codons, culminating in NMD of the transcript. Unlike previous approaches, our technique enabled observation of alternative TL variants for hundreds of genes and revealed significant differences in translation in genes with distinct TL isoforms. TL-seq and TATLseq are useful tools for annotation and functional characterization of TLs, and can be applied to any eukaryotic system to investigate TL-mediated regulation of gene expression.

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Identification of putative regulatory upstream ORFs in the yeast genome using heuristics and evolutionary conservation

Cited by 46 publications

References 40 publications

Upstream Open Reading Frames Differentially Regulate Gene-specific Translation in the Integrated Stress Response

Upstream Open Reading Frames Differentially Regulate Gene-specific Translation in the Integrated Stress Response

Conserved non-AUG uORFs revealed by a novel regression analysis of ribosome profiling data

Roles for transcript leaders in translation and mRNA decay revealed by transcript leader sequencing

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