Human NMD ensues independently of stable ribosome stalling

Karousis, Evangelos D.; Gurzeler, Lukas-Adrian; Annibaldis, Giuditta; Dreos, René; Mühlemann, Oliver

doi:10.1101/2019.12.11.872861

Cited by 5 publications

(9 citation statements)

References 54 publications

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“…3΄UTR construct as described before 15 . Amplification of the vector using the primers 5΄-AAT AAG AGC TCC TGC CTC GAG CTT CCT CATC-3΄ and 5΄-AAT AAC ATA TGG TGA TGC TAT TGC TTT ATT TGT AAC-3΄ was followed by restriction digestion with SacI and NdeI and ligation with a 6xMS2-containing insert that was PCR-amplified using the primers 5΄-AAT AAC ATA TGG TTC CCT AAG TCC AAC TAC CAA A-3΄ and 5΄-AAT AAA GAG CTC CCA GAG GTT GAT TGT CGA CC-3΄…”

Section: Preparation Of Viral 5'-utr Mrna and Reporter Rluc Mrnamentioning

confidence: 99%

“…Preparation of in vitro transcribed mRNAs was performed as described 15 Capping buffer (New England Biolabs). The capping reaction was carried out at 37 °C for 1 h and quenched by the addition of acidic P.C.I., followed by RNA purification.…”

Section: In Vitro Transcription Of Reporter Mrnasmentioning

confidence: 99%

“…In vitro translation reactions were performed similarly as described before 15 Additionally, before precipitation, samples were taken for analysis on agarose gels (0.06% bleach, 1% (w/v) agarose).…”

Section: In Vitro Translation Assaysmentioning

confidence: 99%

“…We further investigated how wild type (WT) Nsp1 affects translation of a Renilla Luciferase-encoding reporter mRNA (RLuc) in an S3 HeLa lysate in vitro translation system 15 ( Fig. 3d).…”

mentioning

confidence: 99%

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SARS-CoV-2 Nsp1 binds ribosomal mRNA channel to inhibit translation

Schubert

Karousis

Jomaa

et al. 2020

Preprint

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AbstractThe non-structural protein 1 (Nsp1), also referred to as the host shutoff factor, is the first viral protein that is synthesized in SARS-CoV-2 infected human cells to suppress host innate immune functions1,2. By combining cryo-electron microscopy and biochemical experiments, we show that SARS-CoV-2 Nsp1 binds to the human 40S subunit in ribosomal complexes including the 43S pre-initiation complex. The protein inserts its C-terminal domain at the entrance to the mRNA channel where it interferes with mRNA binding. We observe potent translation inhibition in the presence of Nsp1 in lysates from human cells. Based on the high-resolution structure of the 40S-Nsp1 complex, we identify residues of Nsp1 crucial for mediating translation inhibition. We further show that the full-length 5’ untranslated region of the genomic viral mRNA stimulates translation in vitro, suggesting that SARS-CoV-2 combines inhibition of translation by Nsp1 with efficient translation of the viral mRNA to achieve expression of viral genes3.

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Section: Preparation Of Viral 5'-utr Mrna and Reporter Rluc Mrnamentioning

confidence: 99%

Section: In Vitro Transcription Of Reporter Mrnasmentioning

confidence: 99%

Section: In Vitro Translation Assaysmentioning

confidence: 99%

“…We further investigated how wild type (WT) Nsp1 affects translation of a Renilla Luciferase-encoding reporter mRNA (RLuc) in an S3 HeLa lysate in vitro translation system 15 ( Fig. 3d).…”

mentioning

confidence: 99%

See 2 more Smart Citations

SARS-CoV-2 Nsp1 binds ribosomal mRNA channel to inhibit translation

Schubert

Karousis

Jomaa

et al. 2020

Preprint

Self Cite

154

264

View full text Add to dashboard Cite

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“…Later studies seemed to confirm the idea of slow and inefficient translation termination and ribosome recycling at PTCs due to the absence of a termination promoting 3′UTR mRNP structure that probably includes PABP [ 39 , 41 , 44 , 70 , 71 , 134 , 142 ]. However, according to a recent report, in vitro translation of reporter mRNAs followed by toeprinting in human cell lysates failed to detect a difference in ribosomal occupancy at the termination codons of reporter mRNAs with or without NMD-inducing 3′UTRs and with or without a Poly(A) tail [ 143 ]. These findings were corroborated by ribosomal profiling of endogenous NMD-sensitive and insensitive transcripts in HeLa cells, which revealed that ribosome occupancy at termination codons is similar in both types of transcripts.…”

Section: Is Termination At a Ptc Really Slower Than At An Ntc?mentioning

confidence: 99%

UPF1-Mediated RNA Decay—Danse Macabre in a Cloud

Lavysh

Neu‐Yilik

2020

Biomolecules

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Nonsense-mediated RNA decay (NMD) is the prototype example of a whole family of RNA decay pathways that unfold around a common central effector protein called UPF1. While NMD in yeast appears to be a linear pathway, NMD in higher eukaryotes is a multifaceted phenomenon with high variability with respect to substrate RNAs, degradation efficiency, effector proteins and decay-triggering RNA features. Despite increasing knowledge of the mechanistic details, it seems ever more difficult to define NMD and to clearly distinguish it from a growing list of other UPF1-mediated RNA decay pathways (UMDs). With a focus on mammalian NMD, we here critically examine the prevailing NMD models and the gaps and inconsistencies in these models. By exploring the minimal requirements for NMD and other UMDs, we try to elucidate whether they are separate and definable pathways, or rather variations of the same phenomenon. Finally, we suggest that the operating principle of the UPF1-mediated decay family could be considered similar to that of a computing cloud providing a flexible infrastructure with rapid elasticity and dynamic access according to specific user needs.

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High-throughput 5’P sequencing enables the study of degradation-associated ribosome stalls

Zhang

2020

Preprint

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RNA degradation is critical for gene expression and mRNA quality control. mRNA degradation is intimately connected to the translation process up to the degree that 5'-3' co-translational mRNA degradation can be used as a proxy for ribosome dynamics. Here we present an improved high-throughput 5'P RNA sequencing method (HT-5Pseq). HT-5Pseq is easy, scalable and uses affordable duplex-specific nuclease based rRNA depletion. We investigate in vivo ribosome stalls in Saccharomyces cerevisiae and Schizosaccharomyces pombe focusing on translation termination. We show that degradation intermediates of mRNAs with high termination pausing often use TGA as stop codon preceded by Lys and have relatively shorter poly(A) tails. We investigate the regulation of this process after glucose deprivation and reveal that, in addition to translation initiation, glucose starvation also leads to ribosome stall at the stop codon. Finally, we investigate gene-specific termination pausing across multiple conditions and show that it is a highly regulated process that crosstalks with codon optimality and the length of poly(A) remaining after deadenylation. In summary, HT-5Pseq is an improved and cost-effective approach that allows to investigate the crosstalk between RNA degradation and the translation process and that is particularly useful to examine its coupling at the level of translation termination. Key points:• HT-5Pseq facilitates high throughput investigation of 5'P mRNA degradation intermediates.• Gene-specific ribosome pause at termination level is common and environmentally regulated.• Ribosome stall at termination level are associated with TGA usage and shorter deadenylated poly(A) tails.

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Human NMD ensues independently of stable ribosome stalling

Cited by 5 publications

References 54 publications

SARS-CoV-2 Nsp1 binds ribosomal mRNA channel to inhibit translation

SARS-CoV-2 Nsp1 binds ribosomal mRNA channel to inhibit translation

UPF1-Mediated RNA Decay—Danse Macabre in a Cloud

High-throughput 5’P sequencing enables the study of degradation-associated ribosome stalls

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