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 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 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 Regulation of Natural mRNAs by the Nonsense-Mediated mRNA Decay Pathway

2014 ◽  
Vol 13 (9) ◽  
pp. 1126-1135 ◽  
Author(s):  
Megan Peccarelli ◽  
Bessie W. Kebaara
Keyword(s):  
Gene Expression ◽  
Mrna Decay ◽  
Regulation Of Gene Expression ◽  
Degradation Pathway ◽  
Mrna Degradation ◽  
Content Type ◽  
Mrna Regulation ◽  
Premature Termination Codons ◽  
Nonsense Mediated Mrna Decay ◽  
Eukaryotic Organisms

ABSTRACT The nonsense-mediated mRNA decay (NMD) pathway is a specialized mRNA degradation pathway that degrades select mRNAs. This pathway is conserved in all eukaryotes examined so far, and it triggers the degradation of mRNAs that prematurely terminate translation. Originally identified as a pathway that degrades mRNAs with premature termination codons as a result of errors during transcription, splicing, or damage to the mRNA, NMD is now also recognized as a pathway that degrades some natural mRNAs. The degradation of natural mRNAs by NMD has been identified in multiple eukaryotes, including Saccharomyces cerevisiae , Drosophila melanogaster , Arabidopsis thaliana , and humans. S. cerevisiae is used extensively as a model to study natural mRNA regulation by NMD. Inactivation of the NMD pathway in S. cerevisiae affects approximately 10% of the transcriptome. Similar percentages of natural mRNAs in the D. melanogaster and human transcriptomes are also sensitive to the pathway, indicating that NMD is important for the regulation of gene expression in multiple organisms. NMD can either directly or indirectly regulate the decay rate of natural mRNAs. Direct NMD targets possess NMD-inducing features. This minireview focuses on the regulation of natural mRNAs by the NMD pathway, as well as the features demonstrated to target these mRNAs for decay by the pathway in S. cerevisiae . We also compare NMD-targeting features identified in S. cerevisiae with known NMD-targeting features in other eukaryotic organisms.

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 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 Regulation of Multiple Core Spliceosomal Proteins by Alternative Splicing-Coupled Nonsense-Mediated mRNA Decay

2008 ◽  
Vol 28 (13) ◽  
pp. 4320-4330 ◽  
Author(s):  
Arneet L. Saltzman ◽  
Yoon Ki Kim ◽  
Qun Pan ◽  
Matthew M. Fagnani ◽  
Lynne E. Maquat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Mrna Decay ◽  
Splice Variants ◽  
Cellular Functions ◽  
Regulate Gene Expression ◽  
Premature Termination Codons ◽  
Microarray Profiling ◽  
Nonsense Mediated Mrna Decay ◽  
Regulate Gene

ABSTRACT Alternative splicing (AS) can regulate gene expression by introducing premature termination codons (PTCs) into spliced mRNA that subsequently elicit transcript degradation by the nonsense-mediated mRNA decay (NMD) pathway. However, the range of cellular functions controlled by this process and the factors required are poorly understood. By quantitative AS microarray profiling, we find that there are significant overlaps among the sets of PTC-introducing AS events affected by individual knockdown of the three core human NMD factors, Up-Frameshift 1 (UPF1), UPF2, and UPF3X/B. However, the levels of some PTC-containing splice variants are less or not detectably affected by the knockdown of UPF2 and/or UPF3X, compared with the knockdown of UPF1. The intron sequences flanking the affected alternative exons are often highly conserved, suggesting important regulatory roles for these AS events. The corresponding genes represent diverse cellular functions, and surprisingly, many encode core spliceosomal proteins and assembly factors. We further show that conserved, PTC-introducing AS events are enriched in genes that encode core spliceosomal proteins. Where tested, altering the expression levels of these core spliceosomal components affects the regulation of PTC-containing splice variants from the corresponding genes. Together, our results show that AS-coupled NMD can have different UPF factor requirements and is likely to regulate many general components of the spliceosome. The results further implicate general spliceosomal components in AS regulation.

scholarly journals VHLFrameshift Mutation as Target of Nonsense-Mediated mRNA Decay inDrosophila melanogasterand Human HEK293 Cell Line

2009 ◽  
Vol 2009 ◽  
pp. 1-9 ◽  
Author(s):  
Lucia Micale ◽  
Lucia Anna Muscarella ◽  
Marco Marzulli ◽  
Bartolomeo Augello ◽  
Patrizia Tritto ◽  
...  
Keyword(s):  
Mrna Decay ◽  
Phenotypic Variability ◽  
Familial Cancer ◽  
Cancer Syndrome ◽  
Hek 293 ◽  
Von Hippel Lindau ◽  
Premature Termination Codons ◽  
Exon 1 ◽  
Nonsense Mediated Mrna Decay ◽  
Human Mutation

There are many well-studied examples of human phenotypes resulting from nonsense or frameshift mutations that are modulated by Nonsense-Mediated mRNA Decay (NMD), a process that typically degrades transcripts containing premature termination codons (PTCs) in order to prevent translation of unnecessary or aberrant transcripts. Different types of germline mutations in theVHLgene cause the von Hippel-Lindau disease, a dominantly inherited familial cancer syndrome with a marked phenotypic variability and age-dependent penetrance. By generating theDrosophilaUAS:Upf1D45Bline we showed the possible involvement of NMD mechanism in the modulation of the c.172delG frameshift mutation located in the exon 1 ofVhlgene. Further, by Quantitative Real-time PCR (QPCR) we demonstrated that the corresponding c.163delG human mutation is targeted by NMD in human HEK 293 cells. The UAS:Upf1D45Bline represents a useful system to identify novel substrates of NMD pathway inDrosophila melanogaster. Finally, we suggest the possible role of NMD on the regulation ofVHLmutations.

scholarly journals Mechanisms and Regulation of Nonsense-Mediated mRNA Decay and Nonsense-Associated Altered Splicing in Lymphoid Cells

2020 ◽  
Author(s):  
Jean-Marie Lambert ◽  
Mohamad Omar Ashi ◽  
Nivine Srour ◽  
Laurent Delpy ◽  
Jérôme Saulière
Keyword(s):  
Quality Control ◽  
Mrna Decay ◽  
Dominant Negative ◽  
Lymphoid Cells ◽  
Surveillance Systems ◽  
Rna Surveillance ◽  
Accelerated Degradation ◽  
Premature Termination Codons ◽  
Rna Quality Control ◽  
Nonsense Mediated Mrna Decay

The presence of premature termination codons (PTCs) in transcripts is dangerous for the cell as they encode potentially deleterious truncated proteins that can act with dominant-negative or gain-of-function effects. To avoid synthesis of these shortened polypeptides, several RNA surveillance systems can be activated to decrease the level of PTC-containing mRNAs. Nonsense-mediated mRNA decay (NMD) ensures an accelerated degradation of mRNAs harboring PTCs by using several key NMD factors such as up-frameshift (UPF) proteins. Another pathway called nonsense-associated altered splicing (NAS) upregulates transcripts that have skipped disturbing PTCs by alternative splicing. Therefore, these RNA quality control processes eliminate abnormal PTC-containing mRNAs from the cells by using positive and negative responses. In this review, we will describe the general mechanisms of NMD and NAS and their respective involvement in the decay of aberrant immunoglobulin and TCR transcripts in lymphoid cells.

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