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.

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 Suppression of aminoglycoside-induced premature termination codon readthrough by the TRP channel inhibitor AC1903

2021 ◽  
Author(s):  
Alireza Baradaran-Heravi ◽  
Claudia C Bauer ◽  
Isabelle B Pickles ◽  
Sara Hosseini-Farahabadi ◽  
Aruna Balgi ◽  
...  
Keyword(s):  
Premature Termination ◽  
Genetic Disorders ◽  
Premature Termination Codon ◽  
Cation Channels ◽  
Trpc Channels ◽  
Nonsense Mutations ◽  
Variable Effect ◽  
Premature Termination Codons ◽  
Ic50 Values ◽  
Nonsense Mediated Mrna Decay

Nonsense mutations, which occur in ~11% of patients with genetic disorders, introduce premature termination codons (PTCs) that lead to truncated proteins and promote nonsense-mediated mRNA decay. Aminoglycosides such as gentamicin and G418 permit PTC readthrough and so may address this problem. However, their effects are variable between patients, making clinical use of aminoglycosides challenging. In this study, we addressed the hypothesis that TRP non-selective cation channels contribute to the variable effect of aminoglycosides by controlling their cellular uptake. To attempt to identify the channel type involved, we tested AC1903, a 2-aminobenzimidazole derivative recently reported to selectively inhibit TRPC5 cation channels. AC1903 consistently suppressed G418 uptake and G418-induced PTC readthrough in the DMS-114 cell line and patient-derived JEB01 keratinocytes. In an effort to validate the suggested role of TRPC5, we tested an independent and more potent inhibitor called Pico145, which affects channels containing TRPC1, TRPC4 and TRPC5 but not other TRPCs or other channels. Unexpectedly, Pico145 was completely without effect, suggesting that AC1903 may work through other or additional targets. Consistent with this suggestion, AC1903 inhibited multiple TRPC channels including homomeric TRPC3, TRPC4, TRPC5, TRPC6 as well as concatemeric TRPC4-C1 and TRPC5-C1 channels, all with low micromolar IC50 values. It also inhibited TRPV4 channels but had weak or no effects on TRPV1 and no effect on another non-selective cation channel, PIEZO1. Overall, our study reveals a suppressor of aminoglycoside-mediated PTC readthrough (i.e., AC1903) but suggests that this compound has previously unrecognised effects. These effects require further investigation to determine the molecular mechanism by which AC1903 suppresses aminoglycoside uptake and PTC readthrough.

scholarly journals Genetic screens identify connections between ribosome recycling and nonsense mediated decay

2021 ◽  
Author(s):  
Karole N D'Orazio ◽  
Laura N. Lessen ◽  
Anthony J. Veltri ◽  
Zachary Neiman ◽  
Miguel E. Pacheco ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Premature Termination ◽  
Stop Codon ◽  
Premature Termination Codon ◽  
Termination Codon ◽  
Genetic Screens ◽  
Regulatory Pathway ◽  
Nonsense Mediated Decay ◽  
Stop Codons ◽  
Reported Data

The decay of messenger RNA with a premature termination codon (PTC) by nonsense mediated decay (NMD) is an important regulatory pathway for eukaryotes and an essential pathway in mammals. NMD is typically triggered by the ribosome terminating at a stop codon that is aberrantly distant from the poly-A tail. Here, we use a fluorescence screen to identify factors involved in NMD in S. cerevisiae . In addition to the known NMD factors, including the entire UPF family (UPF1, UPF2 and UPF3), as well as NMD4 and EBS1 , we identify factors known to function in post-termination recycling and characterize their contribution to NMD. We then use a series of modified reporter constructs that block both elongating and scanning ribosomes downstream of stop codons and demonstrate that a deficiency in recycling of 80S ribosomes or 40S subunits stabilizes NMD substrates. These observations in S. cerevisiae expand on recently reported data in mammals indicating that the 60S recycling factor ABCE1 is important for NMD (1,2) by showing that increased activities of both elongating and scanning ribosomes (80S or 40S) in the 3’UTR correlate with a loss of NMD.

scholarly journals Nonsense-Mediated mRNA Decay Mutation in Aspergillus nidulans

2006 ◽  
Vol 5 (11) ◽  
pp. 1838-1846 ◽  
Author(s):  
Igor Y. Morozov ◽  
Susana Negrete-Urtasun ◽  
Joan Tilburn ◽  
Christine A. Jansen ◽  
Mark X. Caddick ◽  
...  
Keyword(s):  
Aspergillus Nidulans ◽  
Mrna Decay ◽  
Premature Termination ◽  
Regulation Of Gene Expression ◽  
Premature Termination Codon ◽  
Termination Codon ◽  
Urate Oxidase ◽  
Genes Encoding ◽  
Nonsense Mediated Mrna Decay ◽  
Nonsense Codons

ABSTRACT An Aspergillus nidulans mutation, designated nmdA1, has been selected as a partial suppressor of a frameshift mutation and shown to truncate the homologue of the Saccharomyces cerevisiae nonsense-mediated mRNA decay (NMD) surveillance component Nmd2p/Upf2p. nmdA1 elevates steady-state levels of premature termination codon-containing transcripts, as demonstrated using mutations in genes encoding xanthine dehydrogenase (hxA), urate oxidase (uaZ), the transcription factor mediating regulation of gene expression by ambient pH (pacC), and a protease involved in pH signal transduction (palB). nmdA1 can also stabilize pre-mRNA (unspliced) and wild-type transcripts of certain genes. Certain premature termination codon-containing transcripts which escape NMD are relatively stable, a feature more in common with certain nonsense codon-containing mammalian transcripts than with those in S. cerevisiae. As in S. cerevisiae, 5′ nonsense codons are more effective at triggering NMD than 3′ nonsense codons. Unlike the mammalian situation but in common with S. cerevisiae and other lower eukaryotes, A. nidulans is apparently impervious to the position of premature termination codons with respect to the 3′ exon-exon junction.

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 Metabolic stress promotes stop-codon readthrough and phenotypic heterogeneity

2020 ◽  
Vol 117 (36) ◽  
pp. 22167-22172
Author(s):  
Hong Zhang ◽  
Zhihui Lyu ◽  
Yongqiang Fan ◽  
Christopher R. Evans ◽  
Karl W. Barber ◽  
...  
Keyword(s):  
Phenotypic Diversity ◽  
Stop Codon ◽  
Single Cells ◽  
Metabolic Stress ◽  
Aminoglycoside Antibiotic ◽  
Bacterial Cells ◽  
Excess Carbon ◽  
Stop Codons ◽  
Release Factors ◽  
Stop Codon Readthrough

Accurate protein synthesis is a tightly controlled biological process with multiple quality control steps safeguarded by aminoacyl-transfer RNA (tRNA) synthetases and the ribosome. Reduced translational accuracy leads to various physiological changes in both prokaryotes and eukaryotes. Termination of translation is signaled by stop codons and catalyzed by release factors. Occasionally, stop codons can be suppressed by near-cognate aminoacyl-tRNAs, resulting in protein variants with extended C termini. We have recently shown that stop-codon readthrough is heterogeneous among single bacterial cells. However, little is known about how environmental factors affect the level and heterogeneity of stop-codon readthrough. In this study, we have combined dual-fluorescence reporters, mass spectrometry, mathematical modeling, and single-cell approaches to demonstrate that a metabolic stress caused by excess carbon substantially increases both the level and heterogeneity of stop-codon readthrough. Excess carbon leads to accumulation of acid metabolites, which lower the pH and the activity of release factors to promote readthrough. Furthermore, our time-lapse microscopy experiments show that single cells with high readthrough levels are more adapted to severe acid stress conditions and are more sensitive to an aminoglycoside antibiotic. Our work thus reveals a metabolic stress that promotes translational heterogeneity and phenotypic diversity.

scholarly journals Premature termination codons in SOD1 causing Amyotrophic Lateral Sclerosis are predicted to escape the nonsense-mediated mRNA decay

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Claire Guissart ◽  
Kevin Mouzat ◽  
Jovana Kantar ◽  
Baptiste Louveau ◽  
Paul Vilquin ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Mrna Decay ◽  
Premature Termination ◽  
Underlying Mechanism ◽  
Missense Mutations ◽  
Gene Encoding ◽  
Premature Termination Codons ◽  
Nonsense Mediated Mrna Decay ◽  
Inherited Mutations ◽  
Lateral Sclerosis

AbstractAmyotrophic lateral sclerosis (ALS) is the most common and severe adult-onset motoneuron disease and has currently no effective therapy. Approximately 20% of familial ALS cases are caused by dominantly-inherited mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1), which represents one of the most frequent genetic cause of ALS. Despite the overwhelming majority of ALS-causing missense mutations in SOD1, a minority of premature termination codons (PTCs) have been identified. mRNA harboring PTCs are known to be rapidly degraded by nonsense-mediated mRNA decay (NMD), which limits the production of truncated proteins. The rules of NMD surveillance varying with PTC location in mRNA, we analyzed the localization of PTCs in SOD1 mRNA to evaluate whether or not those PTCs can be triggered to degradation by the NMD pathway. Our study shows that all pathogenic PTCs described in SOD1 so far can theoretically escape the NMD, resulting in the production of truncated protein. This finding supports the hypothesis that haploinsufficiency is not an underlying mechanism of SOD1 mutant-associated ALS and suggests that PTCs found in the regions that trigger NMD are not pathogenic. Such a consideration is particularly important since the availability of SOD1 antisense strategies, in view of variant treatment assignment.

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