scholarly journals Cellular variability of nonsense-mediated mRNA decay

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hanae Sato ◽  
Robert H. Singer
Keyword(s):  
Single Cell ◽  
Mrna Decay ◽  
Translation Termination ◽  
Single Cells ◽  
Mrna Degradation ◽  
Globin Genes ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Translational Readthrough ◽  
Nonsense Mediated Mrna Decay

AbstractNonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that eliminates transcripts containing premature termination codons (PTCs). Half-lives of the mRNAs containing PTCs demonstrate that a small percent escape surveillance and do not degrade. It is not known whether this escape represents variable mRNA degradation within cells or, alternatively cells within the population are resistant. Here we demonstrate a single-cell approach with a bi-directional reporter, which expresses two β-globin genes with or without a PTC in the same cell, to characterize the efficiency of NMD in individual cells. We found a broad range of NMD efficiency in the population; some cells degraded essentially all of the mRNAs, while others escaped NMD almost completely. Characterization of NMD efficiency together with NMD regulators in single cells showed cell-to-cell variability of NMD reflects the differential level of surveillance factors, SMG1 and phosphorylated UPF1. A single-cell fluorescent reporter system that enabled detection of NMD using flow cytometry revealed that this escape occurred either by translational readthrough at the PTC or by a failure of mRNA degradation after successful translation termination at the PTC.

scholarly journals Cellular variability of nonsense-mediated mRNA decay

2021 ◽  
Author(s):  
Hanae Sato ◽  
Robert H Singer
Keyword(s):  
Single Cell ◽  
Mrna Decay ◽  
Translation Termination ◽  
Single Cells ◽  
Mrna Degradation ◽  
Globin Genes ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Translational Readthrough ◽  
Nonsense Mediated Mrna Decay

Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that eliminates transcripts containing premature termination codons (PTCs). Half-lives of the mRNAs containing PTCs demonstrate that a small percent escape surveillance and do not degrade. It is not known whether this escape represents variable mRNA degradation within cells or, alternatively cells within the population are resistant. Here we demonstrate a single-cell approach with a bi-directional reporter, which expresses two β-globin genes with or without a PTC in the same cell, to characterize the efficiency of NMD in individual cells. We found a broad range of NMD efficiency in the population; some cells degraded essentially all of the mRNAs, while others escaped NMD almost completely. Characterization of NMD efficiency together with NMD regulators in single cells showed cell-to-cell variability of NMD reflects the differential level of surveillance factors, SMG1 and phosphorylated UPF1. A single-cell fluorescent reporter system that enabled detection of NMD using flow cytometry revealed that this escape occurred either by translational readthrough at the PTC or by a failure of mRNA degradation after successful translation termination at the PTC.

scholarly journals Single-cell detection of Nonsense-Mediated mRNA decay

2021 ◽  
Author(s):  
Robert Singer ◽  
Hanae Sato
Keyword(s):  
Single Cell ◽  
Mrna Decay ◽  
Single Cells ◽  
Mrna Degradation ◽  
Cell Detection ◽  
Globin Genes ◽  
Reporter System ◽  
Single Cell Detection ◽  
Translational Readthrough ◽  
Nonsense Mediated Mrna Decay

Abstract Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that eliminates transcripts containing premature termination codons (PTCs). Half-lives of the mRNAs containing PTCs demonstrates that a small percent escape surveillance and do not degrade. It is not known whether this escape represents variable mRNA degradation within cells or, alternatively cells within the population are resistant. Here we demonstrate a single-cell approach with a bi-directional reporter, which expresses two b-globin genes with or without a PTC in the same cell, to characterize the efficiency of NMD in individual cells. We found a broad range of NMD efficiency in the population; some cells degraded essentially all of the mRNAs, while others escaped NMD almost completely. Characterization of NMD efficiency together with NMD regulators in single cells showed cell-to-cell variability of NMD reflects the differential level of surveillance factors, SMG1 and phosphorylated UPF1. A single-cell fluorescent reporter system that enabled detection of NMD using flow cytometry revealed that this escape occurred either by translational readthrough at the PTC or by failure of mRNA degradation after successful translation termination at the PTC.

scholarly journals Upf1p, Nmd2p, and Upf3p Regulate the Decapping and Exonucleolytic Degradation of both Nonsense-Containing mRNAs and Wild-Type mRNAs

2001 ◽  
Vol 21 (5) ◽  
pp. 1515-1530 ◽  
Author(s):  
Feng He ◽  
Allan Jacobson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mrna Decay ◽  
Translation Termination ◽  
Mrna Degradation ◽  
Wild Type ◽  
Rapid Degradation ◽  
Yeast Strains ◽  
Nonsense Mediated Mrna Decay ◽  
Exonucleolytic Degradation ◽  
Decapping Enzyme

ABSTRACT In Saccharomyces cerevisiae, rapid degradation of nonsense-containing mRNAs requires the decapping enzyme Dcp1p, the 5′-to-3′ exoribonuclease Xrn1p, and the three nonsense-mediated mRNA decay (NMD) factors, Upf1p, Nmd2p, and Upf3p. To identify specific functions for the NMD factors, we analyzed the mRNA decay phenotypes of yeast strains containing deletions of DCP1 orXRN1 and UPF1, NMD2, or UPF3. Our results indicate that Upf1p, Nmd2p, and Upf3p regulate decapping and exonucleolytic degradation of nonsense-containing mRNAs. In addition, we show that these factors also regulate the same processes in the degradation of wild-type mRNAs. The participation of the NMD factors in general mRNA degradation suggests that they may regulate an aspect of translation termination common to all transcripts.

scholarly journals Nonsense-mediated mRNA decay utilizes complementary mechanisms to suppress mRNA and protein accumulation

2021 ◽  
Author(s):  
Dylan B. Udy ◽  
Robert K Bradley
Keyword(s):  
Mrna Decay ◽  
Mrna Degradation ◽  
Protein Accumulation ◽  
Safe Harbor ◽  
Reporter System ◽  
Truncated Protein ◽  
Protein Levels ◽  
Premature Termination Codons ◽  
Nonsense Mediated Mrna Decay ◽  
Accurate Quantification

Nonsense-mediated mRNA decay (NMD) is an essential, highly conserved quality control pathway that detects and degrades mRNAs containing premature termination codons (PTCs). Although the essentiality of NMD is frequently ascribed to its prevention of truncated protein accumulation, the extent to which NMD actually suppresses proteins encoded by NMD-sensitive transcripts is less well-understood than NMD-mediated suppression of mRNA. Here, we describe a reporter system that permits accurate quantification of both mRNA and protein levels via stable integration of paired reporters encoding NMD-sensitive and -insensitive transcripts into the AAVS1 safe harbor loci in human cells. We use this system to demonstrate that NMD suppresses proteins encoded by NMD-sensitive transcripts by up to ~8-fold more than the mRNA itself. Our data indicate that NMD limits the accumulation of proteins encoded by NMD substrates by mechanisms beyond mRNA degradation, such that even when NMD-sensitive mRNAs escape destruction, their encoded proteins are still effectively suppressed.

scholarly journals Nonsense-mediated mRNA decay relies on “two-factor authentication” by SMG5-SMG7

2020 ◽  
Author(s):  
Volker Boehm ◽  
Sabrina Kueckelmann ◽  
Jennifer V. Gerbracht ◽  
Thiago Britto-Borges ◽  
Janine Altmüller ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Decay ◽  
Translation Termination ◽  
Mrna Degradation ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene ◽  
Nonsense Mediated Mrna Decay ◽  
Functional Hierarchy ◽  
Improved Model ◽  
Dependent Pathway

AbstractEukaryotic gene expression is constantly regulated and controlled by the translation-coupled nonsense-mediated mRNA decay (NMD) pathway. Aberrant translation termination leads to NMD activation and robust clearance of NMD targets via two seemingly independent and redundant mRNA degradation branches. Here, we uncover that the loss of the first SMG5-SMG7-dependent pathway also inactivates the second SMG6-dependent branch, indicating an unexpected functional hierarchy of the final NMD steps. Transcriptome-wide analyses of SMG5-SMG7-depleted cells confirm complete NMD inhibition resulting in massive transcriptomic alterations. The NMD activity conferred by SMG5-SMG7 is determined to varying degrees by their interaction with the central NMD factor UPF1, heterodimer formation and the initiation of deadenylation. Surprisingly, we find that SMG5 functionally substitutes SMG7 and vice versa. Our data support an improved model for NMD execution that requires two-factor authentication involving UPF1 phosphorylation and SMG5-SMG7 recruitment to access SMG6 activity.

scholarly journals SMG5-SMG7 authorize nonsense-mediated mRNA decay by enabling SMG6 endonucleolytic activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Volker Boehm ◽  
Sabrina Kueckelmann ◽  
Jennifer V. Gerbracht ◽  
Sebastian Kallabis ◽  
Thiago Britto-Borges ◽  
...  
Keyword(s):  
Mrna Decay ◽  
Translation Termination ◽  
Mrna Degradation ◽  
Functional Connection ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene ◽  
Nonsense Mediated Mrna Decay ◽  
Improved Model ◽  
Dependent Pathway

AbstractEukaryotic gene expression is constantly controlled by the translation-coupled nonsense-mediated mRNA decay (NMD) pathway. Aberrant translation termination leads to NMD activation, resulting in phosphorylation of the central NMD factor UPF1 and robust clearance of NMD targets via two seemingly independent and redundant mRNA degradation branches. Here, we uncover that the loss of the first SMG5-SMG7-dependent pathway also inactivates the second SMG6-dependent branch, indicating an unexpected functional connection between the final NMD steps. Transcriptome-wide analyses of SMG5-SMG7-depleted cells confirm exhaustive NMD inhibition resulting in massive transcriptomic alterations. Intriguingly, we find that the functionally underestimated SMG5 can substitute the role of SMG7 and individually activate NMD. Furthermore, the presence of either SMG5 or SMG7 is sufficient to support SMG6-mediated endonucleolysis of NMD targets. Our data support an improved model for NMD execution that features two-factor authentication involving UPF1 phosphorylation and SMG5-SMG7 recruitment to access SMG6 activity.

scholarly journals Nonsense-mediated mRNA decay uses complementary mechanisms to suppress mRNA and protein accumulation

2021 ◽  
Vol 5 (3) ◽  
pp. e202101217
Author(s):  
Dylan B Udy ◽  
Robert K Bradley
Keyword(s):  
Mrna Decay ◽  
Mrna Degradation ◽  
Protein Accumulation ◽  
Safe Harbor ◽  
Reporter System ◽  
Truncated Protein ◽  
Protein Levels ◽  
Premature Termination Codons ◽  
Nonsense Mediated Mrna Decay ◽  
Accurate Quantification

Nonsense-mediated mRNA decay (NMD) is an essential, highly conserved quality control pathway that detects and degrades mRNAs containing premature termination codons. Although the essentiality of NMD is frequently ascribed to its prevention of truncated protein accumulation, the extent to which NMD actually suppresses proteins encoded by NMD-sensitive transcripts is less well-understood than NMD-mediated suppression of mRNA. Here, we describe a reporter system that permits accurate quantification of both mRNA and protein levels via stable integration of paired reporters encoding NMD-sensitive and NMD-insensitive transcripts into the AAVS1 safe harbor loci in human cells. We use this system to demonstrate that NMD suppresses proteins encoded by NMD-sensitive transcripts by up to eightfold more than the mRNA itself. Our data indicate that NMD limits the accumulation of proteins encoded by NMD substrates by mechanisms beyond mRNA degradation, such that even when NMD-sensitive mRNAs escape destruction, their encoded proteins are still effectively suppressed.

scholarly journals A UPF1 variant drives conditional remodeling of nonsense-mediated mRNA decay

2021 ◽  
Author(s):  
Sarah E. Fritz ◽  
Soumya Ranganathan ◽  
J. Robert Hogg
Keyword(s):  
Gene Expression ◽  
Mrna Decay ◽  
Gene Expression Regulation ◽  
Translation Termination ◽  
Translational Repression ◽  
The Core ◽  
Human Genes ◽  
Rna Quality Control ◽  
Nonsense Mediated Mrna Decay

AbstractThe nonsense-mediated mRNA decay (NMD) pathway monitors translation termination to degrade transcripts with premature stop codons and regulate thousands of human genes. Due to the major role of NMD in RNA quality control and gene expression regulation, it is important to understand how the pathway responds to changing cellular conditions. Here we show that an alternative mammalian-specific isoform of the core NMD factor UPF1, termed UPF1LL, enables condition-dependent remodeling of NMD specificity. UPF1LL associates more stably with potential NMD target mRNAs than the major UPF1SL isoform, expanding the scope of NMD to include many transcripts normally immune to the pathway. Unexpectedly, the enhanced persistence of UPF1LL on mRNAs supports induction of NMD in response to rare translation termination events. Thus, while canonical NMD is abolished by translational repression, UPF1LL activity is enhanced, providing a mechanism to rapidly rewire NMD specificity in response to cellular stress.

scholarly journals Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Nicolas Lemus-Diaz ◽  
Kai O. Böker ◽  
Ignacio Rodriguez-Polo ◽  
Michael Mitter ◽  
Jasmin Preis ◽  
...  
Keyword(s):  
Single Cell ◽  
Mirna Gene ◽  
Gene Repression ◽  
Single Cell Level ◽  
Reporter System ◽  
Cell Level ◽  
Fluorescent Reporter

scholarly journals Tpa1p Is Part of an mRNP Complex That Influences Translation Termination, mRNA Deadenylation, and mRNA Turnover in Saccharomyces cerevisiae

2006 ◽  
Vol 26 (14) ◽  
pp. 5237-5248 ◽  
Author(s):  
Kim M. Keeling ◽  
Joe Salas-Marco ◽  
Lev Z. Osherovich ◽  
David M. Bedwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mrna Decay ◽  
Potential Role ◽  
Translation Termination ◽  
Tail Length ◽  
Fold Increase ◽  
Reading Frame ◽  
Yeast Strains ◽  
Messenger Ribonucleoprotein ◽  
Nonsense Mediated Mrna Decay

ABSTRACT In this report, we show that the Saccharomyces cerevisiae protein Tpa1p (for termination and polyadenylation) influences translation termination efficiency, mRNA poly(A) tail length, and mRNA stability. Tpa1p is encoded by the previously uncharacterized open reading frame YER049W. Yeast strains carrying a deletion of the TPA1 gene (tpa1Δ) exhibited increased readthrough of stop codons, and coimmunoprecipitation assays revealed that Tpa1p interacts with the translation termination factors eRF1 and eRF3. In addition, the tpa1Δ mutation led to a 1.5- to 2-fold increase in the half-lives of mRNAs degraded by the general 5′→3′ pathway or the 3′→5′ nonstop decay pathway. In contrast, this mutation did not have any affect on the nonsense-mediated mRNA decay pathway. Examination of mRNA poly(A) tail length revealed that poly(A) tails are longer than normal in a tpa1Δ strain. Consistent with a potential role in regulating poly(A) tail length, Tpa1p was also found to coimmunoprecipitate with the yeast poly(A) binding protein Pab1p. These results suggest that Tpa1p is a component of a messenger ribonucleoprotein complex bound to the 3′ untranslated region of mRNAs that affects translation termination, deadenylation, and mRNA decay.

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