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 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 Interrogating the degradation pathways of unstable mRNAs with XRN1-resistant sequences

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Volker Boehm ◽  
Jennifer V. Gerbracht ◽  
Marie-Charlotte Marx ◽  
Niels H. Gehring
Keyword(s):  
Gene Expression ◽  
Mrna Decay ◽  
Mrna Degradation ◽  
Eukaryotic Cells ◽  
Mrna Turnover ◽  
Degradation Pathways ◽  
Messenger Rnas ◽  
Endonucleolytic Cleavage ◽  
Nonsense Mediated Mrna Decay ◽  
Cytokine Mrnas

Abstract The turnover of messenger RNAs (mRNAs) is a key regulatory step of gene expression in eukaryotic cells. Due to the complexity of the mammalian degradation machinery, the contribution of decay factors to the directionality of mRNA decay is poorly understood. Here we characterize a molecular tool to interrogate mRNA turnover via the detection of XRN1-resistant decay fragments (xrFrag). Using nonsense-mediated mRNA decay (NMD) as a model pathway, we establish xrFrag analysis as a robust indicator of accelerated 5′–3′ mRNA decay. In tethering assays, monitoring xrFrag accumulation allows to distinguish decapping and endocleavage activities from deadenylation. Moreover, xrFrag analysis of mRNA degradation induced by miRNAs, AU-rich elements (AREs) as well as the 3′ UTRs of cytokine mRNAs reveals the contribution of 5′–3′ decay and endonucleolytic cleavage. Our work uncovers formerly unrecognized modes of mRNA turnover and establishes xrFrag as a powerful tool for RNA decay analyses.

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 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.

Recognition of nonsense mRNA: towards a unified model

2008 ◽  
Vol 36 (3) ◽  
pp. 497-501 ◽  
Author(s):  
Oliver Mühlemann
Keyword(s):  
Gene Expression ◽  
Present Article ◽  
Protein Complex ◽  
Mrna Decay ◽  
Translation Termination ◽  
Physical Distance ◽  
Unified Model ◽  
Nonsense Mediated Mrna Decay ◽  
Correct Translation ◽  
Premature Translation Termination

Among the different cellular surveillance mechanisms that ensure accurate gene expression, nonsense-mediated mRNA decay rapidly degrades mRNAs harbouring PTCs (premature translation-termination codons) and thereby prevents the accumulation of potentially deleterious proteins with C-terminal truncations. In the present article, I review recent data from yeast, fluitflies, nematode worms and human cells and endeavour to merge these results into a unified model for recognition of nonsense mRNA. According to this model, the distinction between translation termination at PTCs and at ‘normal’ termination codons relies on the physical distance between the terminating ribosome and PABP [poly(A)-binding protein]. Correct translation termination is promoted by a PABP-mediated signal to the terminating ribosome, whereas the absence of this signal leads to the assembly of an mRNA decay-promoting protein complex including the conserved NMD factors UPF (up-frameshift) 1–3.

Role of small nuclear RNAs in eukaryotic gene expression

2013 ◽  
Vol 54 ◽  
pp. 79-90 ◽  
Author(s):  
Saba Valadkhan ◽  
Lalith S. Gunawardane
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Rna Stability ◽  
Splice Sites ◽  
Branch Site ◽  
Small Nuclear Rnas ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene ◽  
Rna Biogenesis

Eukaryotic cells contain small, highly abundant, nuclear-localized non-coding RNAs [snRNAs (small nuclear RNAs)] which play important roles in splicing of introns from primary genomic transcripts. Through a combination of RNA–RNA and RNA–protein interactions, two of the snRNPs, U1 and U2, recognize the splice sites and the branch site of introns. A complex remodelling of RNA–RNA and protein-based interactions follows, resulting in the assembly of catalytically competent spliceosomes, in which the snRNAs and their bound proteins play central roles. This process involves formation of extensive base-pairing interactions between U2 and U6, U6 and the 5′ splice site, and U5 and the exonic sequences immediately adjacent to the 5′ and 3′ splice sites. Thus RNA–RNA interactions involving U2, U5 and U6 help position the reacting groups of the first and second steps of splicing. In addition, U6 is also thought to participate in formation of the spliceosomal active site. Furthermore, emerging evidence suggests additional roles for snRNAs in regulation of various aspects of RNA biogenesis, from transcription to polyadenylation and RNA stability. These snRNP-mediated regulatory roles probably serve to ensure the co-ordination of the different processes involved in biogenesis of RNAs and point to the central importance of snRNAs in eukaryotic gene expression.

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