scholarly journals Ribosome recycling factor ABCE1 depletion inhibits nonsense-mediated mRNA decay by promoting stop codon readthrough

2019 ◽  
Author(s):  
Giuditta Annibaldis ◽  
René Dreos ◽  
Michal Domanski ◽  
Sarah Carl ◽  
Oliver Mühlemann
Keyword(s):  
Mrna Decay ◽  
Stop Codon ◽  
Translation Termination ◽  
Ribosome Profiling ◽  
List Type ◽  
Ribosome Recycling Factor ◽  
Stop Codons ◽  
Nonsense Mediated Mrna Decay ◽  
Ribosome Disassembly ◽  
Stop Codon Readthrough

SUMMARYNonsense-mediated mRNA decay (NMD) is an essential post-transcriptional surveillance pathway in vertebrates that appears to be mechanistically linked with translation termination. To gain more insight into this connection, we interfered with translation termination by depleting human cells of the ribosome recycling factor ABCE1, which resulted in an upregulation of many but not all endogenous NMD-sensitive mRNAs. Notably, the suppression of NMD on these mRNAs occurs at a step prior to their SMG6-mediated endonucleolytic cleavage. Ribosome profiling revealed that ABCE1 depletion results in ribosome stalling at stop codons and increased ribosome occupancy in 3’ UTRs, indicative of enhanced stop codon readthrough or re-initiation. Using reporter genes, we further demonstrate that the absence of ABCE1 indeed increases the rate of readthrough, which would explain the observed NMD inhibition, since enhanced readthrough has been previously shown to render NMD-sensitive transcripts resistant to NMD by displacing NMD triggering factors like UPF1 and exon junction complexes (EJCs) from the 3’ UTR. Collectively, our results show that improper ribosome disassembly interferes with proper NMD activation.HighlightsABCE1 knockdown suppresses NMD of many NMD-sensitive mRNAsThe observed NMD inhibition occurs at a stage prior to SMG6-mediated cleavage of the mRNAABCE1 depletion enhances ribosome occupancy at stop codons and in the 3’ UTRABCE1 depletion enhances readthrough of the stop codonEnhanced readthrough inhibits NMD, presumably by clearing the 3’ UTR of NMD factors

scholarly journals Destabilization of Eukaryote mRNAs by 5′ Proximal Stop Codons Can Occur Independently of the Nonsense-Mediated mRNA Decay Pathway

Cells ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 800 ◽  
Author(s):  
Barbara Gorgoni ◽  
Yun-Bo Zhao ◽  
J. Krishnan ◽  
Ian Stansfield
Keyword(s):  
Mrna Stability ◽  
Mrna Decay ◽  
Closed Loop ◽  
Stop Codon ◽  
Translation Termination ◽  
Translation System ◽  
Translation Elongation ◽  
Reading Frame ◽  
Stop Codons ◽  
Nonsense Mediated Mrna Decay

In eukaryotes, the binding of poly(A) binding protein (PAB) to the poly(A) tail is central to maintaining mRNA stability. PABP interacts with the translation termination apparatus, and with eIF4G to maintain 3′–5′ mRNA interactions as part of an mRNA closed loop. It is however unclear how ribosome recycling on a closed loop mRNA is influenced by the proximity of the stop codon to the poly(A) tail, and how post-termination ribosome recycling affects mRNA stability. We show that in a yeast disabled for nonsense mediated mRNA decay (NMD), a PGK1 mRNA with an early stop codon at codon 22 of the reading frame is still highly unstable, and that this instability cannot be significantly countered even when 50% stop codon readthrough is triggered. In an NMD-deficient mutant yeast, stable reporter alleles with more 3′ proximal stop codons could not be rendered unstable through Rli1-depletion, inferring defective Rli1 ribosome recycling is insufficient in itself to trigger mRNA instability. Mathematical modelling of a translation system including the effect of ribosome recycling and poly(A) tail shortening supports the hypothesis that impaired ribosome recycling from 5′ proximal stop codons may compromise initiation processes and thus destabilize the mRNA. A model is proposed wherein ribosomes undergo a maturation process during early elongation steps, and acquire competency to re-initiate on the same mRNA as translation elongation progresses beyond the very 5′ proximal regions of the mRNA.

scholarly journals Polypyrimidine tract binding protein 1 protects mRNAs from recognition by the nonsense-mediated mRNA decay pathway

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Zhiyun Ge ◽  
Bao Lin Quek ◽  
Karen L Beemon ◽  
J Robert Hogg
Keyword(s):  
Gene Expression ◽  
Mrna Decay ◽  
Gene Expression Regulation ◽  
Stop Codon ◽  
Binding Protein ◽  
Polypyrimidine Tract ◽  
Polypyrimidine Tract Binding ◽  
Stop Codons ◽  
Polypyrimidine Tract Binding Protein ◽  
Nonsense Mediated Mrna Decay

The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs containing long 3'UTRs to perform dual roles in mRNA quality control and gene expression regulation. However, expansion of vertebrate 3'UTR functions has required a physical expansion of 3'UTR lengths, complicating the process of detecting nonsense mutations. We show that the polypyrimidine tract binding protein 1 (PTBP1) shields specific retroviral and cellular transcripts from NMD. When bound near a stop codon, PTBP1 blocks the NMD protein UPF1 from binding 3'UTRs. PTBP1 can thus mark specific stop codons as genuine, preserving both the ability of NMD to accurately detect aberrant mRNAs and the capacity of long 3'UTRs to regulate gene expression. Illustrating the wide scope of this mechanism, we use RNA-seq and transcriptome-wide analysis of PTBP1 binding sites to show that many human mRNAs are protected by PTBP1 and that PTBP1 enrichment near stop codons correlates with 3'UTR length and resistance to NMD.

scholarly journals New insights into the interplay between the translation machinery and nonsense-mediated mRNA decay factors

2018 ◽  
Vol 46 (3) ◽  
pp. 503-512 ◽  
Author(s):  
Etienne Raimondeau ◽  
Joshua C. Bufton ◽  
Christiane Schaffitzel
Keyword(s):  
Mrna Decay ◽  
Stop Codon ◽  
Translation Termination ◽  
Premature Stop Codon ◽  
Molecular Function ◽  
Translation Machinery ◽  
New Findings ◽  
Nascent Chain ◽  
Nonsense Mediated Mrna Decay

Faulty mRNAs with a premature stop codon (PTC) are recognized and degraded by nonsense-mediated mRNA decay (NMD). Recognition of a nonsense mRNA depends on translation and on the presence of NMD-enhancing or the absence of NMD-inhibiting factors in the 3′-untranslated region. Our review summarizes our current understanding of the molecular function of the conserved NMD factors UPF3B and UPF1, and of the anti-NMD factor Poly(A)-binding protein, and their interactions with ribosomes translating PTC-containing mRNAs. Our recent discovery that UPF3B interferes with human translation termination and enhances ribosome dissociation in vitro, whereas UPF1 is inactive in these assays, suggests a re-interpretation of previous experiments and modification of prevalent NMD models. Moreover, we discuss recent work suggesting new functions of the key NMD factor UPF1 in ribosome recycling, inhibition of translation re-initiation and nascent chain ubiquitylation. These new findings suggest that the interplay of UPF proteins with the translation machinery is more intricate than previously appreciated, and that this interplay quality-controls the efficiency of termination, ribosome recycling and translation re-initiation.

scholarly journals Readthrough of stop codons under limiting ABCE1 concentration involves frameshifting and inhibits nonsense-mediated mRNA decay

2020 ◽  
Vol 48 (18) ◽  
pp. 10259-10279
Author(s):  
Giuditta Annibaldis ◽  
Michal Domanski ◽  
René Dreos ◽  
Lara Contu ◽  
Sarah Carl ◽  
...  
Keyword(s):  
Quality Control ◽  
Mrna Decay ◽  
Translation Termination ◽  
Human Cells ◽  
Ribosome Recycling Factor ◽  
Ribosome Stalling ◽  
Endonucleolytic Cleavage ◽  
Nonsense Mediated Mrna Decay ◽  
Insight Into ◽  
Reading Frames

Abstract To gain insight into the mechanistic link between translation termination and nonsense-mediated mRNA decay (NMD), we depleted the ribosome recycling factor ABCE1 in human cells, resulting in an upregulation of NMD-sensitive mRNAs. Suppression of NMD on these mRNAs occurs prior to their SMG6-mediated endonucleolytic cleavage. ABCE1 depletion caused ribosome stalling at termination codons (TCs) and increased ribosome occupancy in 3′ UTRs, implying enhanced TC readthrough. ABCE1 knockdown indeed increased the rate of readthrough and continuation of translation in different reading frames, providing a possible explanation for the observed NMD inhibition, since enhanced readthrough displaces NMD activating proteins from the 3′ UTR. Our results indicate that stalling at TCs triggers ribosome collisions and activates ribosome quality control. Collectively, we show that improper translation termination can lead to readthrough of the TC, presumably due to ribosome collisions pushing the stalled ribosomes into the 3′ UTR, where it can resume translation in-frame as well as out-of-frame.

scholarly journals Nonsense mRNA suppression via nonstop decay

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Joshua A Arribere ◽  
Andrew Z Fire
Keyword(s):  
Mrna Decay ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Nonsense Mediated Decay ◽  
Stop Codons ◽  
Nonstop Mrna Decay ◽  
Rna Fragments ◽  
Nonsense Mediated Mrna Decay ◽  
Key Steps

Nonsense-mediated mRNA decay is the process by which mRNAs bearing premature stop codons are recognized and cleared from the cell. While considerable information has accumulated regarding recognition of the premature stop codon, less is known about the ensuing mRNA suppression. During the characterization of a second, distinct translational surveillance pathway (nonstop mRNA decay), we trapped intermediates in nonsense mRNA degradation. We present data in support of a model wherein nonsense-mediated decay funnels into the nonstop decay pathway in Caenorhabditis elegans. Specifically, our results point to SKI-exosome decay and pelota-based ribosome removal as key steps facilitating suppression and clearance of prematurely-terminated translation complexes. These results suggest a model in which premature stop codons elicit nucleolytic cleavage, with the nonstop pathway disengaging ribosomes and degrading the resultant RNA fragments to suppress ongoing expression.

scholarly journals Stop Codon Context Influences Genome-Wide Stimulation of Termination Codon Readthrough by Aminoglycosides

2019 ◽  
Author(s):  
Jamie R Wangen ◽  
Rachel Green
Keyword(s):  
Mammalian Cells ◽  
Stop Codon ◽  
Translation Termination ◽  
Premature Termination Codon ◽  
Ribosome Profiling ◽  
Termination Codon ◽  
Stop Codons ◽  
Genome Wide ◽  
A Genome

AbstractStop codon readthrough (SCR) occurs when the ribosome miscodes at a stop codon. Such readthrough events can be therapeutically desirable when a premature termination codon (PTC) is found in a critical gene. To study SCR in vivo in a genome-wide manner, we treated mammalian cells with aminoglycosides and performed ribosome profiling. We find that in addition to stimulating readthrough of PTCs, aminoglycosides stimulate readthrough of normal termination codons (NTCs) genome-wide. Stop codon identity, the nucleotide following the stop codon, and the surrounding mRNA sequence context all influence the likelihood of SCR. In comparison to NTCs, downstream stop codons in 3′UTRs are recognized less efficiently by ribosomes, suggesting that targeting of critical stop codons for readthrough may be achievable without general disruption of translation termination. Finally, we find that G418 treatment globally alters gene expression with substantial effects on translation of histone genes, selenoprotein genes, and S-adenosylmethionine decarboxylase (AMD1).

scholarly journals Eukaryotic ribosome recognizes 3 context and stimulates stop codon readthrough

2021 ◽  
Author(s):  
Elizaveta Sokolova ◽  
Tatiana Egorova ◽  
Alexey Shuvalov ◽  
Elena Alkalaeva
Keyword(s):  
18S Rrna ◽  
Stop Codon ◽  
Translation Termination ◽  
Translation System ◽  
Stop Codons ◽  
Nucleotide Context ◽  
The One ◽  
A Site ◽  
Stop Codon Readthrough ◽  
Cognate Trna

It is known that the nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3 contexts have been described that are unfavourable for translation termination; however, the exact molecular mechanism that mediates their effect remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3 stop codon contexts. Our results revealed that ribosomes can independently recognize certain contexts and ignore stop codons that are followed by these sequences. Moreover, the efficiency of translation termination at the weak 3 contexts was almost equal to the one at the standard context. We propose that weak 3 contexts interact with the 18S rRNA provoking a conformational change in the U-turn-like structure of the stop codon in the A site of ribosome. This change makes incorporation of the near-cognate tRNA more preferable than recognition of the stop codon by the release factors and increases readthrough.

scholarly journals Stop codon context influences genome-wide stimulation of termination codon readthrough by aminoglycosides

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jamie R Wangen ◽  
Rachel Green
Keyword(s):  
Mammalian Cells ◽  
Stop Codon ◽  
Translation Termination ◽  
Premature Termination Codon ◽  
Ribosome Profiling ◽  
Termination Codon ◽  
Stop Codons ◽  
Genome Wide ◽  
A Genome

Stop codon readthrough (SCR) occurs when the ribosome miscodes at a stop codon. Such readthrough events can be therapeutically desirable when a premature termination codon (PTC) is found in a critical gene. To study SCR in vivo in a genome-wide manner, we treated mammalian cells with aminoglycosides and performed ribosome profiling. We find that in addition to stimulating readthrough of PTCs, aminoglycosides stimulate readthrough of normal termination codons (NTCs) genome-wide. Stop codon identity, the nucleotide following the stop codon, and the surrounding mRNA sequence context all influence the likelihood of SCR. In comparison to NTCs, downstream stop codons in 3′UTRs are recognized less efficiently by ribosomes, suggesting that targeting of critical stop codons for readthrough may be achievable without general disruption of translation termination. Finally, we find that G418-induced miscoding alters gene expression with substantial effects on translation of histone genes, selenoprotein genes, and S-adenosylmethionine decarboxylase (AMD1).

scholarly journals Premature termination codon readthrough in Drosophila varies in a developmental and tissue-specific manner

2019 ◽  
Author(s):  
Yanan Chen ◽  
Tianhui Sun ◽  
Zhuo Bi ◽  
Jian-Quan Ni ◽  
Jose C. Pastor-Pareja ◽  
...  
Keyword(s):  
Life Cycle ◽  
Premature Termination ◽  
Stop Codon ◽  
Premature Termination Codon ◽  
Anatomical Site ◽  
Stop Codons ◽  
Premature Termination Codons ◽  
Nonsense Mediated Mrna Decay ◽  
Rescue Mechanism ◽  
Stop Codon Readthrough

AbstractDespite their essential function in terminating translation, readthrough of stop codons occurs more frequently than previously supposed. However, little is known about the regulation of stop codon readthrough by anatomical site and over the life cycle of animals. Here, we developed a set of reporters to measure readthrough in Drosophila melanogaster. A focused RNAi screen in whole animals identified upf1 as a mediator of readthrough, suggesting that the stop codons in the reporters were recognized as premature termination codons (PTCs). We found readthrough rates of PTCs varied significantly throughout the life cycle of flies, being highest in older adult flies. Furthermore, readthrough rates varied dramatically by tissue and, intriguingly, were highest in fly brains, specifically neurons and not glia. This was not due to differences in reporter abundance or nonsense-mediated mRNA decay (NMD) surveillance between these tissues. Overall, our data reveal temporal and spatial variation of PTC-mediated readthrough in animals, and suggest that readthrough may be a potential rescue mechanism for PTC-harboring transcripts when the NMD surveillance pathway is inhibited.

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