Genome-wide search for novel human uORFs and N-terminal protein extensions using ribosomal footprinting

Fritsch, Claudia; Herrmann, Alexander; Nothnagel, Michael; Szafranski, Karol; Huse, Klaus; Schumann, Frank; Schreiber, Stefan; Platzer, Matthias; Krawczak, Michael; Hampe, Jochen; Brosch, Mario

doi:10.1101/gr.139568.112

Cited by 207 publications

(192 citation statements)

References 68 publications

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“…Recent ribosome profiling studies have illustrated that previously uncharacterized uORFs with non-canonical initiation codons are more commonly translated than previously thought (3,4,6). This observation has been followed by the characterization of multiple mechanisms involving bypass of uORFs due to their non-canonical initiation codons that regulate gene-specific translation during cellular stress conditions (4,26,68).…”

Section: Ribosome Bypass and Non-canonical Start Codonsmentioning

confidence: 99%

“…This finding indicates that those uORFs that are retained throughout species likely maintain functional significance. This idea is emphasized in genome-wide analyses of uORF conservation in which the presence of an uORF and the regulatory nature of uORF(s) is suggested to be conserved throughout species (6,7,(85)(86)(87).…”

Section: Evolutionary Conservation Of Uorf-mediated Translation Mechamentioning

confidence: 99%

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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: Ribosome Bypass and Non-canonical Start Codonsmentioning

confidence: 99%

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

View full text Add to dashboard Cite

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“…Prior to the development of Ribo-Seq, translated uORFs with regulatory functions were often identified by mutant screening or evolutionary conservation (Hill and Morris, 1993;Delbecq et al, 1994;Wiese et al, 2004;Imai et al, 2006;Hayden and Jorgensen, 2007). Although Ribo-Seq data have been used in the identification of translated uORFs (Fritsch et al, 2012;Liu et al, 2013;Ingolia et al, 2014), the application of Ribo-Seq data to the identification of ribosome stalling uORFs has not been reported. In this study, we show that some CPuORFs overaccumulate 59-truncated RNA ends with a signature of stacked ribosomes (Figure 2A).…”

Section: Investigation Of Regulatory Uorfs Using the Rna Degradomementioning

confidence: 99%

Global Analysis of Truncated RNA Ends Reveals New Insights into Ribosome Stalling in Plants

et al. 2016

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High-throughput approaches for profiling the 59 ends of RNA degradation intermediates on a genome-wide scale are frequently applied to analyze and validate cleavage sites guided by microRNAs (miRNAs). However, the complexity of the RNA degradome other than miRNA targets is currently largely uncharacterized, and this limits the application of RNA degradome studies. We conducted a global analysis of 59-truncated mRNA ends that mapped to coding sequences (CDSs) of Arabidopsis thaliana, rice (Oryza sativa), and soybean (Glycine max). Based on this analysis, we provide multiple lines of evidence to show that the plant RNA degradome contains in vivo ribosome-protected mRNA fragments. We observed a 3-nucleotide periodicity in the position of free 59 RNA ends and a bias toward the translational frame. By examining conserved peptide upstream open reading frames (uORFs) of Arabidopsis and rice, we found a predominance of 59 termini of RNA degradation intermediates that were separated by a length equal to a ribosome-protected mRNA fragment. Through the analysis of RNA degradome data, we discovered uORFs and CDS regions potentially associated with stacked ribosomes in Arabidopsis. Furthermore, our analysis of RNA degradome data suggested that the binding of Arabidopsis ARGONAUTE7 to a noncleavable target site of miR390 might directly hinder ribosome movement. This work demonstrates an alternative use of RNA degradome data in the study of ribosome stalling.

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“…Recently, a global approach to discover uORF and NH 2 -terminally extended proteins using ribosomal footprinting in a monocytic cell line (THP-1) showed that about half of the annotated coding sequences have uORF that might have an effect on their expression (18). The same technique was employed to assess the effects of oxidative stress on translation in yeast (21).…”

Section: Discussionmentioning

confidence: 99%

Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants

Tsotakos

Silveyra

Lin

et al. 2015

American Journal of Physiology-Lung Cellular and Molecular Phys

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Surfactant protein A (SP-A), a molecule with roles in lung innate immunity and surfactant-related functions, is encoded by two genes in humans: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The mRNAs from these genes differ in their 5′-untranslated regions (5′-UTR) due to differential splicing. The 5′-UTR variant ACD′ is exclusively found in transcripts of SP-A1, but not in those of SP-A2. Its unique exon C contains two upstream AUG codons (uAUGs) that may affect SP-A1 translation efficiency. The first uAUG (u1) is in frame with the primary start codon (p), but the second one (u2) is not. The purpose of this study was to assess the impact of uAUGs on SP-A1 expression. We employed RT-qPCR to determine the presence of exon C-containing SP-A1 transcripts in human RNA samples. We also used in vitro techniques including mutagenesis, reporter assays, and toeprinting analysis, as well as in silico analyses to determine the role of uAUGs. Exon C-containing mRNA is present in most human lung tissue samples and its expression can, under certain conditions, be regulated by factors such as dexamethasone or endotoxin. Mutating uAUGs resulted in increased luciferase activity. The mature protein size was not affected by the uAUGs, as shown by a combination of toeprint and in silico analysis for Kozak sequence, secondary structure, and signal peptide and in vitro translation in the presence of microsomes. In conclusion, alternative splicing may introduce uAUGs in SP-A1 transcripts, which in turn negatively affect SP-A1 translation, possibly affecting SP-A1/SP-A2 ratio, with potential for clinical implication.

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Genome-wide search for novel human uORFs and N-terminal protein extensions using ribosomal footprinting

Cited by 207 publications

References 68 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

Global Analysis of Truncated RNA Ends Reveals New Insights into Ribosome Stalling in Plants

Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants

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