Analysis of Nonsense‐Mediated mRNA Decay in Mammalian Cells

Nicholson, Pamela; Joncourt, Raphael; Mühlemann, Oliver

doi:10.1002/0471143030.cb2704s55

Cited by 25 publications

(24 citation statements)

References 114 publications

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“…Cell harvesting for protein samples (derived from the same sample as RNA preparation) and measurement of relative mRNA levels by reverse transcription quantitative polymerase chain reaction (RTqPCR) were done as described in Nicholson et al (2012). Briefly, 2 × 10 5 cell equivalents were analyzed on a 10% PAGE, and detection was performed using Anti-RENT1 (UPF1) (Bethyl, A300-038A), anti-EST1 (SMG6) (Abcam, ab87539), Anti-SMG7 (Bethyl, A302-170A), and Anti-CPSF73 (custom made) antibodies.…”

Section: Experimental Methodsmentioning

confidence: 99%

Transcriptome-wide identification of NMD-targeted human mRNAs reveals extensive redundancy between SMG6- and SMG7-mediated degradation pathways

Colombo

Karousis

Bourquin

et al. 2016

RNA

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173

270

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Besides degrading aberrant mRNAs that harbor a premature translation termination codon (PTC), nonsense-mediated mRNA decay (NMD) also targets many seemingly "normal" mRNAs that encode for full-length proteins. To identify a bona fide set of such endogenous NMD targets in human cells, we applied a meta-analysis approach in which we combined transcriptome profiling of knockdowns and rescues of the three NMD factors UPF1, SMG6, and SMG7. We provide evidence that this combinatorial approach identifies NMD-targeted transcripts more reliably than previous attempts that focused on inactivation of single NMD factors. Our data revealed that SMG6 and SMG7 act on essentially the same transcripts, indicating extensive redundancy between the endo-and exonucleolytic decay routes. Besides mRNAs, we also identified as NMD targets many long noncoding RNAs as well as miRNA and snoRNA host genes. The NMD target feature with the most predictive value is an intron in the 3 ′ UTR, followed by the presence of upstream open reading frames (uORFs) and long 3 ′ UTRs. Furthermore, the 3 ′ UTRs of NMD-targeted transcripts tend to have an increased GC content and to be phylogenetically less conserved when compared to 3 ′ UTRs of NMD insensitive transcripts.

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Section: Experimental Methodsmentioning

confidence: 99%

Transcriptome-wide identification of NMD-targeted human mRNAs reveals extensive redundancy between SMG6- and SMG7-mediated degradation pathways

Colombo

Karousis

Bourquin

et al. 2016

RNA

Self Cite

173

270

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“…pSUPERpuro plasmids used in knockdown experiments of UPF1 (Supplemental Fig. 3), UPF2 + UPF3b, SMG5 + SMG7, SMG6 + SMG7, and a control (scrambled) were generated as described (Brummelkamp et al 2002;Paillusson et al 2005;Nicholson et al 2012); the shRNA target sequences are listed in the Supplemental Material. pSUPERpuro-empty is described in Yepiskoposyan et al (2011).…”

Section: Methodsmentioning

confidence: 99%

“…The cells were harvested 3 d after transfection. For details, see Nicholson et al (2012). Efficiencies of knockdowns were verified on the protein level by Western blotting and/or on the mRNA level by qPCR analysis.…”

Section: Cell Experiments and Rnai-mediated Knockdownsmentioning

confidence: 99%

“…Total RNA was extracted from the cells using guanidium thiocyanate: phenol:chloroform extraction (Nicholson et al 2012) or the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich). The RNA samples were digested with 15-20 units of RNase-free recombinant DNaseI (Roche) for 45 min at 37°C and afterward purified by phenol:chloroform:isoamyl alcohol extraction (25:24:1) and ethanol precipitation according to standard protocols.…”

Section: Reverse Transcription and Quantitative Real-time Pcr (Rt-qpcr)mentioning

confidence: 99%

“…The samples were measured in duplicates using RotorGene 6000 (Corbett) with the following conditions: 3 min at 95°C, 5 sec at 95°C, and 15 sec at 60°C in 40 cycles. Analysis was done according to Nicholson et al (2012).…”

Section: Reverse Transcription and Quantitative Real-time Pcr (Rt-qpcr)mentioning

confidence: 99%

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Comparison of EJC-enhanced and EJC-independent NMD in human cells reveals two partially redundant degradation pathways

Metze

Herzog

Ruepp

et al. 2013

RNA

Self Cite

117

126

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Nonsense-mediated mRNA decay (NMD) is a eukaryotic post-transcriptional gene regulation mechanism that eliminates mRNAs with the termination codon (TC) located in an unfavorable environment for efficient translation termination. The best-studied NMD-targeted mRNAs contain premature termination codons (PTCs); however, NMD regulates even many physiological mRNAs. An exon-junction complex (EJC) located downstream from a TC acts as an NMD-enhancing signal, but is not generally required for NMD. Here, we compared these "EJC-enhanced" and "EJC-independent" modes of NMD with regard to their requirement for seven known NMD factors in human cells using two well-characterized NMD reporter genes (immunoglobulin μ and β-Globin) with or without an intron downstream from the PTC. We show that both NMD modes depend on UPF1 and SMG1, but detected transcript-specific differences with respect to the requirement for UPF2 and UPF3b, consistent with previously reported UPF2-and UPF3-independent branches of NMD. In addition and contrary to expectation, a higher sensitivity of EJC-independent NMD to reduced UPF2 and UPF3b concentrations was observed. Our data further revealed a redundancy of the endo-and exonucleolytic mRNA degradation pathways in both modes of NMD. Moreover, the relative contributions of both decay pathways differed between the reporters, with PTC-containing immunoglobulin μ transcripts being preferentially subjected to SMG6-mediated endonucleolytic cleavage, whereas β-Globin transcripts were predominantly degraded by the SMG5/SMG7-dependent pathway. Overall, the surprising heterogeneity observed with only two NMD reporter pairs suggests the existence of several mechanistically distinct branches of NMD in human cells.

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Nonsense‐mediated mRNA decay: The challenge of telling right from wrong in a complex transcriptome

2019

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The nonsense-mediated mRNA decay pathway selects and degrades its targets using a dense network of RNA-protein and protein-protein interactions. Together, these interactions allow the pathway to collect copious information about the translating mRNA, including translation termination status, splice junction positions, mRNP composition, and 3 0 UTR length and structure. The core NMD machinery, centered on the RNA helicase UPF1, integrates this information to determine the efficiency of decay. A picture of NMD is emerging in which many factors contribute to the dynamics of decay complex assembly and disassembly, thereby influencing the probability of decay. The ability of the NMD pathway to recognize mRNP features of diverse potential substrates allows it to simultaneously perform quality control and regulatory functions. In vertebrates, increased transcriptome complexity requires balance between these two functions since high NMD efficiency is desirable for maintenance of quality control fidelity but may impair expression of normal mRNAs. NMD has adapted to this challenge by employing mechanisms to enhance identification of certain potential substrates, while using sequence-specific RNA-binding proteins to shield others from detection. These elaborations on the conserved NMD mechanism permit more sensitive posttranscriptional gene regulation but can have severe deleterious consequences, including the failure to degrade pathogenic aberrant mRNAs in many B cell lymphomas. This article is categorized under: RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms K E Y W O R D S exon junction complex, messenger ribonucleoprotein complex, nonsense-mediated decay, RNA decay, UPF1

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Analysis of Nonsense‐Mediated mRNA Decay in Mammalian Cells

Cited by 25 publications

References 114 publications

Transcriptome-wide identification of NMD-targeted human mRNAs reveals extensive redundancy between SMG6- and SMG7-mediated degradation pathways

Transcriptome-wide identification of NMD-targeted human mRNAs reveals extensive redundancy between SMG6- and SMG7-mediated degradation pathways

Comparison of EJC-enhanced and EJC-independent NMD in human cells reveals two partially redundant degradation pathways

Nonsense‐mediated mRNA decay: The challenge of telling right from wrong in a complex transcriptome

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