mRNA surveillance: the perfect persist

2002 ◽  
Vol 115 (15) ◽  
pp. 3033-3038 ◽  
Author(s):  
Eileen Wagner ◽  
Jens Lykke-Andersen
Keyword(s):  
Gene Expression ◽  
Premature Termination ◽  
Dominant Negative ◽  
Nonsense Mediated Decay ◽  
Premature Termination Codons ◽  
Mrna Surveillance ◽  
Multistep Process

In eukaryotes, an elaborate set of mechanisms has evolved to ensure that the multistep process of gene expression is accurately executed and adapted to cellular needs. The mRNA surveillance pathway works in this context by assessing the quality of mRNAs to ensure that they are suitable for translation. mRNA surveillance facilitates the detection and destruction of mRNAs that contain premature termination codons by a process called nonsense-mediated decay. Moreover, recent studies have shown that a distinct mRNA surveillance process, called nonstop decay, is responsible for depleting mRNAs that lack in-frame termination codons. mRNA surveillance thereby prevents the synthesis of truncated and otherwise aberrant proteins, which can have dominant-negative and other deleterious effects.

scholarly journals Selective destabilization of polypeptides synthesized from NMD-targeted transcripts

2021 ◽  
Author(s):  
Vincent Chu ◽  
Qing Feng ◽  
Yang Lim ◽  
Sichen Shao
Keyword(s):  
Mammalian Cells ◽  
Premature Termination ◽  
Proteasome Inhibition ◽  
Premature Termination Codon ◽  
Dominant Negative ◽  
Termination Codon ◽  
Negative Effects ◽  
Nonsense Mediated Decay ◽  
Mrna Surveillance ◽  
Reporter Systems

The translation of mRNAs that contain a premature termination codon (PTC) generates truncated proteins that may have toxic dominant negative effects. Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that degrades PTC-containing mRNAs to limit the production of truncated proteins. NMD activation requires a ribosome terminating translation at a PTC, but what happens to the polypeptides synthesized during the translation cycle needed to activate NMD is incompletely understood. Here, by establishing reporter systems that encode the same polypeptide sequence before a normal or premature termination codon, we show that termination of protein synthesis at a PTC is sufficient to selectively destabilize polypeptides in mammalian cells. Proteasome inhibition specifically rescues the levels of nascent polypeptides produced from PTC-containing mRNAs within an hour, but also disrupts mRNA homeostasis within a few hours. PTC-terminated polypeptide destabilization is also alleviated by depleting the central NMD factor UPF1 or SMG1, the kinase that phosphorylates UPF1 to activate NMD, but not by inhibiting SMG1 kinase activity. Our results suggest that polypeptide degradation is linked to PTC recognition in mammalian cells and clarify a framework to investigate these mechanisms.

scholarly journals Premature termination codons in theDMDgene cause reduced local mRNA synthesis

2020 ◽  
Vol 117 (28) ◽  
pp. 16456-16464 ◽  
Author(s):  
Raquel García-Rodríguez ◽  
Monika Hiller ◽  
Laura Jiménez-Gracia ◽  
Zarah van der Pal ◽  
Judit Balog ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Premature Termination ◽  
Epigenetic Mechanism ◽  
Nuclear Compartment ◽  
Transcript Levels ◽  
Nonsense Mediated Decay ◽  
Premature Termination Codons ◽  
Deacetylase Inhibitor ◽  
Repressive Histone Mark

Duchenne muscular dystrophy (DMD) is caused by mutations in theDMDgene leading to the presence of premature termination codons (PTC). Previous transcriptional studies have shown reduced DMD transcript levels in DMD patient and animal model muscles when PTC are present. Nonsense-mediated decay (NMD) has been suggested to be responsible for the observed reduction, but there is no experimental evidence supporting this claim. In this study, we aimed to investigate the mechanism responsible for the drop inDMDexpression levels in the presence of PTC. We observed that the inhibition of NMD does not normalizeDMDgene expression in DMD. Additionally, in situ hybridization showed that DMD messenger RNA primarily localizes in the nuclear compartment, confirming that a cytoplasmic mechanism like NMD indeed cannot be responsible for the observed reduction. Sequencing of nascent RNA to exploreDMDtranscription dynamics revealed a lower rate ofDMDtranscription in patient-derived myotubes compared to healthy controls, suggesting a transcriptional mechanism involved in reduced DMD transcript levels. Chromatin immunoprecipitation in muscle showed increased levels of the repressive histone mark H3K9me3 inmdxmice compared to wild-type mice, indicating a chromatin conformation less prone to transcription inmdxmice. In line with this finding, treatment with the histone deacetylase inhibitor givinostat caused a significant increase in DMD transcript expression inmdxmice. Overall, our findings show that transcription dynamics across theDMDlocus are affected by the presence of PTC, hinting at a possible epigenetic mechanism responsible for this process.

NMD: RNA biology meets human genetic medicine

2010 ◽  
Vol 430 (3) ◽  
pp. 365-377 ◽  
Author(s):  
Madhuri Bhuvanagiri ◽  
Anna M. Schlitter ◽  
Matthias W. Hentze ◽  
Andreas E. Kulozik
Keyword(s):  
Gene Expression ◽  
Global Gene Expression ◽  
The Other ◽  
Surveillance Systems ◽  
Premature Termination Codons ◽  
Rna Biology ◽  
Mrna Surveillance ◽  
Nonsense Mediated Mrna Decay ◽  
The One ◽  
Genetic Medicine

NMD (nonsense-mediated mRNA decay) belongs to the best-studied mRNA surveillance systems of the cell, limiting the synthesis of truncated and potentially harmful proteins on the one hand and playing an initially unexpected role in the regulation of global gene expression on the other hand. In the present review, we briefly discuss the factors involved in NMD, the different models proposed for the recognition of PTCs (premature termination codons), the diverse physiological roles of NMD, the involvement of this surveillance pathway in disease and the current strategies for medical treatment of PTC-related diseases.

scholarly journals Identification of a Human Decapping Complex Associated with hUpf Proteins in Nonsense-Mediated Decay

2002 ◽  
Vol 22 (23) ◽  
pp. 8114-8121 ◽  
Author(s):  
Jens Lykke-Andersen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Premature Termination ◽  
The Other ◽  
Nonsense Mediated Decay ◽  
Cell Extracts ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Premature Termination Codons ◽  
Rate Limiting ◽  
Decapping Enzymes

ABSTRACT Decapping is a key step in general and regulated mRNA decay. In Saccharomyces cerevisiae it constitutes a rate-limiting step in the nonsense-mediated decay pathway that rids cells of mRNAs containing premature termination codons. Here two human decapping enzymes are identified, hDcp1a and hDcp2, as well as a homolog of hDcp1a, termed hDcp1b. Transiently expressed hDcp1a and hDcp2 proteins localize primarily to the cytoplasm and form a complex in human cell extracts. hDcp1a and hDcp2 copurify with decapping activity, an activity sensitive to mutation of critical hDcp residues. Importantly, coimmunoprecipitation assays demonstrate that hDcp1a and hDcp2 interact with the nonsense-mediated decay factor hUpf1, both in the presence and in the absence of the other hUpf proteins, hUpf2, hUpf3a, and hUpf3b. These data suggest that a human decapping complex may be recruited to mRNAs containing premature termination codons by the hUpf proteins.

A global analysis of the reconstitution of PTEN function by translational readthrough of PTEN pathogenic premature termination codons

2021 ◽  
Vol 42 (5) ◽  
pp. 551-566
Author(s):  
Sandra Luna ◽  
Leire Torices ◽  
Janire Mingo ◽  
Laura Amo ◽  
Isabel Rodríguez‐Escudero ◽  
...  
Keyword(s):  
Premature Termination ◽  
Global Analysis ◽  
Premature Termination Codons ◽  
Translational Readthrough

scholarly journals The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Michal Marczyk ◽  
Chunxiao Fu ◽  
Rosanna Lau ◽  
Lili Du ◽  
Alexander J. Trevarton ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Rna Sequencing ◽  
Rna Extraction ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
The Impact ◽  
Formalin Fixed

Abstract Background Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. Methods Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. Results Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63–0.66) and between technical replicates (median expression difference 0.13–0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31–0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91–0.96). Conclusions The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

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