Hypoxic Inhibition of Nonsense-Mediated RNA Decay Regulates Gene Expression and the Integrated Stress Response

Gardner, Lawrence B.

doi:10.1128/mcb.02284-07

Cited by 204 publications

(317 citation statements)

References 48 publications

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“…RNAs associated with tagged UPF1 and GFP proteins were coimmunoprecipitated from cell lysates using anti-Flag antibody, and the relative abundance of various mRNAs in these precipitates was determined. Relative to the GFP IP fraction, the mRNAs of SMG5, SMG6, and SMG7 were substantially enriched (threefold to 3.5-fold) in the UPF1 IP fraction, SMG1, UPF2, and UPF3a mRNAs as well as the mRNA of the well-known NMD substrate MAP3K14 (Mendell et al 2004;Gardner 2008;Stalder and Muhlemann 2009) were moderately enriched (about twofold), and the mRNA levels of UPF3b and the negative control GAPDH were similar in the UPF1 and GFP IP fractions (Fig. 7).…”

Section: Resultsmentioning

confidence: 93%

Autoregulation of the nonsense-mediated mRNA decay pathway in human cells

Yepiskoposyan¹,

Aeschimann²,

Nilsson³

et al. 2011

RNA

219

292

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Nonsense-mediated mRNA decay (NMD) is traditionally portrayed as a quality-control mechanism that degrades mRNAs with truncated open reading frames (ORFs). However, it is meanwhile clear that NMD also contributes to the post-transcriptional gene regulation of numerous physiological mRNAs. To identify endogenous NMD substrate mRNAs and analyze the features that render them sensitive to NMD, we performed transcriptome profiling of human cells depleted of the NMD factors UPF1, SMG6, or SMG7. It revealed that mRNAs up-regulated by NMD abrogation had a greater median 39-UTR length compared with that of the human mRNAome and were also enriched for 39-UTR introns and uORFs. Intriguingly, most mRNAs coding for NMD factors were among the NMD-sensitive transcripts, implying that the NMD process is autoregulated. These mRNAs all possess long 39 UTRs, and some of them harbor uORFs. Using reporter gene assays, we demonstrated that the long 39 UTRs of UPF1, SMG5, and SMG7 mRNAs are the main NMD-inducing features of these mRNAs, suggesting that long 39 UTRs might be a frequent trigger of NMD.

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Section: Resultsmentioning

confidence: 93%

Autoregulation of the nonsense-mediated mRNA decay pathway in human cells

Yepiskoposyan¹,

Aeschimann²,

Nilsson³

et al. 2011

RNA

219

292

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“…Short TL genes present unique post-transcriptional regulatory opportunities for a cell. Their degradation via NMD could be regulated, as conditions such as hypoxia and amino acid starvation have been reported to stabilize NMD substrates in humans (Mendell et al 2004;Gardner 2008). For reporter mRNAs with short TLs, the efficiency of cap-proximal AUG recognition in vitro can be controlled by the levels of eIF1 (Pestova and Kolupaeva 2002), raising the possibility that the levels or activity of eIF1 in vivo may control the production of full-length protein (and the extent of out-of-frame initiation) for short TL genes.…”

Section: Discussionmentioning

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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“…For example, CHOP was identified as a target of the nonsense-mediated mRNA decay pathway that recognizes the presence of a premature termination codon (83). Depletion of the nonsense-mediated mRNA decay machinery from cells results in the stabilization of CHOP mRNA levels (84). CHOP mRNA half-life was also increased more than 2-fold in cells in which the two initiating codons of the CHOP uORF were mutated (14).…”

Section: Other Complexities Of Gene Expression Regulated By Uorfsmentioning

confidence: 99%

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

Young

Wek

2016

Journal of Biological Chemistry

319

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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Hypoxic Inhibition of Nonsense-Mediated RNA Decay Regulates Gene Expression and the Integrated Stress Response

Cited by 204 publications

References 48 publications

Autoregulation of the nonsense-mediated mRNA decay pathway in human cells

Autoregulation of the nonsense-mediated mRNA decay pathway in human cells

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

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

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