Inhibition of nonsense-mediated RNA decay by the tumor microenvironment promotes tumorigenesis
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE30499 | GEO | 2011/07/08
SECONDARY ACCESSION(S): PRJNA143335
REPOSITORIES: GEO
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Project description:Nonsense-mediated RNA decay (NMD) is regulated by a variety of cellular stresses. We expose U2OS cells to several stresses and assess RNA expression in the absence of transcription (i.e. stability). These studies identify transcripts that are stabilized by the physiological inhibition of NMD. U2OS cells rendered hypoxia for three hours, treated with tunicamycin for three hours, treated with emetine for three hours, or depleted of Upf1/Rent1 were treated with DRB, and RNA was collected at 0, 1.5, 3 and 4.5 hrs.
2011-07-08 | E-GEOD-30499 | biostudies-arrayexpress
Project description:Nonsense-mediated mRNA decay (NMD) controls gene expression by eliminating mRNAs with premature or aberrant translation termination. Degradation of NMD substrates is initiated by the central NMD factor UPF1, which recruits the endonuclease SMG6 and the deadenylation-promoting SMG5/7 complex. Here we map transcriptome-wide sites of SMG6-mediated endocleavage via 3′ fragment capture and degradome sequencing.
2017-06-01 | E-MTAB-3625 | biostudies-arrayexpress
Project description:We blocked nonsense-mediated mRNA decay (NMD) pathway and used a custom alternative transcript sensitive Affymetrix microarray to seek alternatively spliced RNAs whose levels increased compared to controls. Keywords: Nonsense Mediated Decay (NMD)
2007-08-31 | GSE6611 | GEO
Project description:Eukaryotic 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 features two-factor authentication involving UPF1 phosphorylation and SMG5-SMG7 recruitment to access SMG6 activity.
2021-05-08 | PXD024747 | Pride
Project description:Nonsense-mediated mRNA decay (NMD) pathway promotes the degradation of several mRNA species, including transcripts containing a premature termination codon. Here,we measured global mRNA half-lives in NMD deficient and wild-type (WT) embryonic stem cells (ESCs). This allowed us to discriminate which transcripts are affected by NMD. Several transcripts had an impaired half-life in Smg5 and Smg6 KO ESCs compared to WT. They belong to different pathways and cellular processing indicating that NMD regulates multiple processes in the cells. Finally, majority of the transcripts had an impairment on the half-life in both Smg5 and Smg6 KO ESCs indicating that endo- and exonucleolityc branches of the NMD pathway target the same mRNAs.
2022-02-02 | GSE153455 | GEO
Project description:Eukaryotic RNAs with premature termination codons (PTCs) are eliminated by nonsense-mediated decay (NMD). While human nonsense RNA degradation can be initiated either by an endonucleolytic cleavage event near the PTC or through decapping, the individual contribution of these activities on endogenous substrates has remained unresolved. Here we unambiguously establish that SMG6-catalyzed endonucleolysis is the primary initiating step in human nonsense RNA decay. We also show that both protein-coding and ‘non-coding’ genes hosting snoRNAs in their introns produce considerable amounts of NMD-sensitive splice variants, suggesting that these RNAs are merely by-products of a primary snoRNA production process. Finally, genes encoding multiple snoRNAs generally yield elevated numbers of alternative transcript isoforms, enabling the differential expression of individual snoRNAs. These findings demonstrate a hitherto unappreciated potential for decoupling of the individual expression levels of functional exon- and intron-encoded species from such composite genes. HEK293 Flp-In T-Rex cells were subjected to siRNA-mediated depletion of XRN1 and co-depletion of XRN1 with either SMG6 or UPF1. All the treated samples together with the controls were subjected to both RNA-seq and 5'-end-seq. RNA-seq was used for detecting NMD isoforms and their expression levels. 5'-end-seq was used for finding NMD decay intermediates (decapped and endocleaved molecules). CAGE was used to distinguish decapped from endocleaved RNA fragments.
2014-11-18 | E-GEOD-57433 | biostudies-arrayexpress
Project description:Premature termination codons (PTCs) lead to loss of protein function by 1) ending mRNA translation before a full-length protein is made, and 2) by triggering nonsense-mediated mRNA decay (NMD), a pathway that targets a PTC-containing mRNA for degradation. Nonsense suppression therapy aims to restore protein function by suppressing termination at PTCs (called readthrough, or RT). However, the efficiency of current readthrough agents is generally quite low. We reasoned that by stimulating both readthrough and increasing mRNA levels, greater protein function can be restored than with readthrough alone. In this study, we developed a series of novel NanoLuc reporters designed to identify and differentiate compounds that induce readthrough, enhance mRNA abundance (by NMD inhibition or other mechanisms), or target both mechanisms simultaneously (referred to as dual RT/NMD reporters). We show that treatments that promote readthrough and NMD inhibition led to synergistic increases in NanoLuc activity of the RT/NMD reporter over either condition alone. We then used a dual RT/NMD reporter to carry out a high throughput screen of a library of 771,345 compounds. We identified 180 compounds with the ability to increase NanoLuc activity in our dual RT/NMD reporter assay. To confirm that such synergy can also amplify protein function in a disease model, we treated cells expressing a CFTR-Y122X mini-intron construct with one hit from this screen, SRI-37240. We found that this compound produced a much larger increase in CFTR activity compared to either the RT compound G418 +/- the NMD inhibitor amlexanox, and the activity obtained could be further enhanced with CFTR modulators. These results confirm the utility of these new reporters to identify many potent new molecules that counteract the effects of nonsense mutations.
2020-12-01 | GSE144140 | GEO
Project description:All eukaryotes studied to date have the capacity to detect and degrade mRNAs harboring premature translation termination codons (PTCs) in a process called nonsense-mediated mRNA decay (NMD) (reviewed in Wagner E & Lykke-Andersen J, 2002). This surveillance system allows the cell to prevent the expression of potentially harmful truncated proteins. trans-acting factors required for NMD in Drosophila are the proteins UPF1, UPF2, UPF3, SMG-1, SMG-5 and SMG-6. To identify mRNAs naturally regulated by NMD, we analyzed expression profiles in Drosophila cells RNAi-depleted of known NMD factors using high-density oligonucleotide arrays.
2005-11-01 | E-MEXP-202 | biostudies-arrayexpress
Project description:Identification of novel, highly penetrant, breast cancer susceptibility genes will require the application of additional strategies beyond that of traditional linkage and candidate gene approaches. Approximately one-third of inherited genetic diseases, including breast cancer susceptibility, are caused by frameshift or nonsense mutations that truncate the protein product [1]. Transcripts harbouring premature termination codons are selectively and rapidly degraded by the nonsense-mediated mRNA decay (NMD) pathway. Blocking the NMD pathway in any given cell will stabilise these mutant transcripts, which can then be detected using gene expression microarrays. This technique, known as gene identification by nonsense-mediated mRNA decay inhibition (GINI), has proved successful in identifying sporadic nonsense mutations involved in many different cancer types. However, the approach has not yet been applied to identify germline mutations involved in breast cancer. We therefore attempted to use GINI on lymphoblastoid cell lines (LCLs) from multiple-case, non-BRCA1/2 breast cancer families in order to identify additional high-risk breast cancer susceptibility genes. We applied GINI to a total of 24 LCLs,established from breast-cancer affected and unaffected women from three multiple-case non-BRCA1/2 breast cancer families. We then used Illumina gene expression microarrays to identify transcripts stabilised by the NMD inhibition. Total RNA obtained from the lymphoblastoid cell lines derived from 24 individuals.
2012-04-11 | E-GEOD-37210 | biostudies-arrayexpress
Project description:Regulation of mRNA stability is a critical step in the control of gene expression. The nonsense mediated mRNA decay (NMD) pathway is one important mRNA stability pathway and has been shown to function in regulating a significant portion of the transcriptome. Perhaps the most significant outstanding question surrounding NMD regulation of endogenous gene expression is the identification of transcripts directly targeted by the NMD pathway versus secondary response genes, whose expression levels are dependent on the activity of direct targets. Here, we present for the first time in an intact animal, the experimental identification of direct targets of NMD. We find that most genes (74%) that are upregulated in NMD mutants appear not to be direct targets of NMD. More surprisingly, we find that the vast majority (97%) of candidate direct targets are not found to be increased in expression in a NMD mutant background. These results imply negative feedback renormalizes the levels of most direct targets of NMD, suggesting they would have been missed in previous analyses. We find that our candidate direct target genes have disproportionately long 3’ UTRs compared to non-targeted control genes. Long 3’ UTRs have been shown to target mRNAs for NMD in experimental systems, and our results imply this is the case in vivo. Our results will allow for the understanding of the function of the NMD pathway in endogenous gene regulation, during normal development, as well as in pathological states. Starting with a Nonsense Mediated Decay(NMD) mutant, in which expression levels of both primary and secondary NMD targets should be increased, we introduce a wild-type copy of the mutated factor and evaluated the transcriptome profile change overtime. Following reintroduction, expression levels of transcripts that are direct targets of NMD should decrease rapidly. Conversely, the levels of indirect targets will remain high until direct targets return to wild-type levels, which will occur later in the timecourse. This allows us to identify direct NMD targets.
2013-07-01 | E-GEOD-47979 | biostudies-arrayexpress