Modulation of nonsense-mediated decay by rapamycin
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE89774 | GEO | 2016/11/11
SECONDARY ACCESSION(S): PRJNA353126
REPOSITORIES: GEO
Similar Datasets
Project description:Cell fate transitions depend on balanced rewiring of transcription and translation programmes to enable ordered developmental progression. We identified a feedback loop between nonsense-mediated mRNA decay (NMD) and translation initiation, in which NMD controls the translation initiation factor Eif4a2 and its premature termination codon encoding isoform (Eif4a2-PTC). This leads to translation of a specific truncated protein, which elicits increased translation rates and is causative for significant delays in mouse embryonic stem cell differentiation. Using immunoprecipitation coupled with mass spectrometry, we identified the interactome of full length Eif4a2 (Eif4a2-iso1) and Eif4a2-PTC. We find that Eif4a2-iso1 is mainly involved in translation initiation, while unstable Eif4a2-PTC shows little interaction with translation initiation factors. Both isoforms can bind to mRNA, as indicated by the interaction with mRNA binding protein, such as NMD factors and Ago2. Finally, Eif4a2-iso1 and Eif4a2-PTC interact with key pluripotency factors, providing a potential explanation for how Eif4a2 controls ESC differentiation.
2022-03-09 | PXD019588 | Pride
Project description:We used a modification of GINI analysis to identify genes containing premature translation termination codons (PTC) generated by nonsense or frameshift mutations in prostate cancer cell lines. The analysis was performed in two steps. In the first step nonsense mediated mRNA decay (NMD) was inhibited in prostate cancer cell lines using incubation with medium containing caffeine for 4 hours. Gene expression analysis of caffeine treated or untreated cells after this step detects mRNA accumulation that takes place for genes containing PTC and as well as for genes that show induction of transciption due to stress caused by NMD inhibition. In the second step either both transcription and NMD or transcription only are blocked by incubating cell in a medium containing either both actinomycin D and caffeine or actinomacin D only for 4 hours. Gene expression analysis after this second step detects mRNA degradation for genes containing PTC as well as for genes that show induction of transciption due to stress caused by NMD inhibition. The efficiency of mRNA degradation for genes containing PTC during this step depends on whether NMD is inhibited or not. The efficiency of mRNA degradation for stress response genes does not depend on whether NMD is inhibited or not. Our modified GINI protocol allows efficient separation of genes containing PTC from the genes that show mRNA increases due to stress response to NMD inhibition.
2009-06-30 | GSE16856 | GEO
Project description:A large number of genetic diseases are caused by various mutations in specific disease genes. A significant proportion (~15%) of these mutations are nonsense mutations that create a premature termination codon (PTC). Consequently, the Nonsense-mediated mRNA Decay (NMD) surveillance pathway degrades a large fraction of PTC-containing mRNA. Translation of the remaining un-degraded PTC-containing mRNA terminates at the PTC, leading to no full-length protein production and hence disease. Therefore, suppressing NMD and translation termination at PTCs becomes an attractive strategy for combating these diseases. This work presents a novel approach, namely targeted PTC pseudouridylation, to suppress nonsense mutations in human cells. By co-transfecting human cells with a PTC-reporter gene and a designer box H/ACA guide RNA gene targeting the PTC, we show that targeted pseudouridylation suppresses both NMD and translation termination at PTCs in the contexts of various disease gene sequences. Furthermore, targeted pseudouridylation exhibits a level of suppression comparable to that of antibiotic treatments, and the suppressive effect is long-lasting in the cell. When targeted pseudouridylation is combined with the antibiotic treatment, a much higher level of suppression is observed. Remarkably, by transfecting a disease model cell line (carrying a chromosomal PTC) with a designer guide RNA gene alone targeting the PTC, we also observe nonsense suppression, suggesting the high potential of this novel approach in treating PTC-associated diseases. Finally, one of the restored full-length proteins is further tested to be functional. Targeted pseudouridylation appears to be the first RNA-directed gene-specific therapeutic approach that suppresses NMD and concurrently promotes PTC read-through.
2022-12-31 | GSE221516 | GEO
Project description:We used a modification of GINI analysis to identify genes containing premature translation termination codons (PTC) generated by nonsense or frameshift mutations in prostate cancer cell lines. The analysis was performed in two steps. In the first step nonsense mediated mRNA decay (NMD) was inhibited in prostate cancer cell lines using incubation with medium containing caffeine for 4 hours. Gene expression analysis of caffeine treated or untreated cells after this step detects mRNA accumulation that takes place for genes containing PTC and as well as for genes that show induction of transciption due to stress caused by NMD inhibition. In the second step either both transcription and NMD or transcription only are blocked by incubating cell in a medium containing either both actinomycin D and caffeine or actinomacin D only for 4 hours. Gene expression analysis after this second step detects mRNA degradation for genes containing PTC as well as for genes that show induction of transciption due to stress caused by NMD inhibition. The efficiency of mRNA degradation for genes containing PTC during this step depends on whether NMD is inhibited or not. The efficiency of mRNA degradation for stress response genes does not depend on whether NMD is inhibited or not. Our modified GINI protocol allows efficient separation of genes containing PTC from the genes that show mRNA increases due to stress response to NMD inhibition. Experiment Overall Design: The LNCaP and 22Rv1 cells were seeded into four 60 mm tissue culture plates each. Caffeine (10 mM) was added to three plates and one plate was incubated without caffeine as a control. Following four hours? incubation, the medium was removed from one plate (with caffeine) and from the control plate and total RNA was prepared using TRIZOL reagent, according to manufacturer?s instructions, and used for an Affymetrix U133Plus2.0 oligonucleotide array hybridization. The caffeine-containing medium of the two remaining plates was removed, the cells were washed twice with phosphate-buffered saline and the medium with actinomycin D (2 mkg/ml) together with caffeine (10 mM) was added to one plate. Actinomycin D alone was added to the other plate. Following another four hours? incubation, the total RNA from both plates was prepared and used for an Affymetrix U133Plus2.0 oligonucleotide array analysis.
2009-07-12 | E-GEOD-16856 | biostudies-arrayexpress
Project description:The first round of translation occurs on mRNAs bound by nuclear cap-binding complex (CBC), which is composed of nuclear cap-binding protein (CBP) 80 and 20. During this round of translation, aberrant mRNAs are recognized and downregulated in abundance by nonsense-mediated mRNA decay (NMD), which is one of the mRNA quality control mechanisms. Here our microarray analysis reveals that the level of cyclin-dependent kinase inhibitor 1A (CDKN1A) mRNAs increases in the cells depleted of cellular NMD factors. Intriguingly, CDKN1A mRNA contains an upstream open reading frame (uORF), which is one of NMD-inducing features. Using chimeric reporter constructs and confocal microscopy, we find that the uORF of CDKN1A mRNA is actively translated and modulates a translational efficiency of the main downstream ORF. Our findings provide the biological insights into the possible role of NMD in diverse pathways mediated by CDKN1A. The microarray analysis performed to analize the cellular NMD substrates that regulated by Upf1, PNRC2 and/or CTIF in HeLa cell. The hypothesis tested in the present study was that endogenous NMD substrates may co-regulated by Upf1, PNRC2 and CTIF. Results provide important information that vast range of cellular NMD substrates are reqired CTIF.
2012-07-23 | GSE37538 | GEO
Project description:The first round of translation occurs on mRNAs bound by nuclear cap-binding complex (CBC), which is composed of nuclear cap-binding protein (CBP) 80 and 20. During this round of translation, aberrant mRNAs are recognized and downregulated in abundance by nonsense-mediated mRNA decay (NMD), which is one of the mRNA quality control mechanisms. Here our microarray analysis reveals that the level of cyclin-dependent kinase inhibitor 1A (CDKN1A) mRNAs increases in the cells depleted of cellular NMD factors. Intriguingly, CDKN1A mRNA contains an upstream open reading frame (uORF), which is one of NMD-inducing features. Using chimeric reporter constructs and confocal microscopy, we find that the uORF of CDKN1A mRNA is actively translated and modulates a translational efficiency of the main downstream ORF. Our findings provide the biological insights into the possible role of NMD in diverse pathways mediated by CDKN1A. The microarray analysis performed to analize the cellular NMD substrates that regulated by Upf1, PNRC2 and/or CTIF in HeLa cell. The hypothesis tested in the present study was that endogenous NMD substrates may co-regulated by Upf1, PNRC2 and CTIF. Results provide important information that vast range of cellular NMD substrates are reqired CTIF. Total RNA obtained from HeLa cells with downregulation of Upf1, PNRC2 or CTIF by siRNA. The up- or down-regulated transcripts were compare to control siRNA treated HeLa cell RNA extract. Significant transcripts were confirmed by replication.
2012-07-22 | E-GEOD-37538 | biostudies-arrayexpress
Project description:Nonsense-mediated mRNA decay (NMD) controls the quality of eukaryotic gene expression and also degrades physiologic mRNAs. How NMD targets are identified is incompletely understood. A central NMD factor is the ATP-dependent RNA helicase UPF1. Neither the distance in space between the termination codon and the poly(A) tail nor the binding of steady-state, largely hypophosphorylated UPF1 is a discriminating marker of cellular NMD targets, unlike for PTC-containing reporter mRNAs when compared to their PTC-free counterparts. Here, we map phosphorylated UPF1 (p-UPF1) binding sites using transcriptome-wide footprinting or DNA oligonucleotide-directed mRNA cleavage to report that p-UPF1 provides the first reliable cellular NMD-target marker. p-UPF1 is enriched on NMD target 3'UTRs along with SMG5 and SMG7 but not SMG1 or SMG6. Immunoprecipitations of UPF1 variants deficient in various aspects of the NMD process in parallel with FRET experiments reveal that ATPase/helicase-deficient UPF1 manifests high levels of RNA binding and disregulated hyperphosphorylation, whereas wild-type UPF1 releases from nonspecific RNA interactions in an ATP hydrolysis-dependent mechanism until an NMD target is identified. 3'UTR-associated UPF1 undergoes regulated phosphorylation on NMD targets, providing a binding platform for mRNA degradative activities. p-UPF1 binding to NMD target 3'UTRs is stabilized by SMG5 and SMG7. Our results help to explain why steady-state UPF1 binding is not a marker for cellular NMD substrates and how this binding is transformed to induce mRNA decay.
2014-08-15 | GSE60045 | GEO
Project description:Nonsense-mediated mRNA decay (NMD) controls the quality of eukaryotic gene expression and also degrades physiologic mRNAs. How NMD targets are identified is incompletely understood. A central NMD factor is the ATP-dependent RNA helicase UPF1. Neither the distance in space between the termination codon and the poly(A) tail nor the binding of steady-state, largely hypophosphorylated UPF1 is a discriminating marker of cellular NMD targets, unlike for PTC-containing reporter mRNAs when compared to their PTC-free counterparts. Here, we map phosphorylated UPF1 (p-UPF1) binding sites using transcriptome-wide footprinting or DNA oligonucleotide-directed mRNA cleavage to report that p-UPF1 provides the first reliable cellular NMD-target marker. p-UPF1 is enriched on NMD target 3'UTRs along with SMG5 and SMG7 but not SMG1 or SMG6. Immunoprecipitations of UPF1 variants deficient in various aspects of the NMD process in parallel with FRET experiments reveal that ATPase/helicase-deficient UPF1 manifests high levels of RNA binding and disregulated hyperphosphorylation, whereas wild-type UPF1 releases from nonspecific RNA interactions in an ATP hydrolysis-dependent mechanism until an NMD target is identified. 3'UTR-associated UPF1 undergoes regulated phosphorylation on NMD targets, providing a binding platform for mRNA degradative activities. p-UPF1 binding to NMD target 3'UTRs is stabilized by SMG5 and SMG7. Our results help to explain why steady-state UPF1 binding is not a marker for cellular NMD substrates and how this binding is transformed to induce mRNA decay. RIP-seq experiments for p-UPF1, control IPs using rabbit IgG and additional control sample without IP were performed in duplicates
2014-08-15 | E-GEOD-60045 | biostudies-arrayexpress
Project description:Background: Alternative splicing (AS), which generates multiple mRNA isoforms from single genes, is crucial for the regulation of eukaryotic gene expression. The flux through competing AS pathways cannot be determined by traditional RNA-Seq, however, because different mRNA isoforms can have widely differing decay rates. Indeed, some mRNA isoforms with extremely short half-lives, such as those subject to translation-dependent nonsense-mediated decay (AS-NMD), may be completely overlooked in even the most extensive RNA-Seq analyses. Results: RNA immunoprecipitation in tandem (RIPiT) of exon junction complex (EJC) components allows for purification of post-splicing mRNA-protein particles (mRNPs) not yet subject to translation (pre-translational mRNPs) and, therefore, translation-dependent mRNA decay. Here we compared EJC RIPiT-Seq to whole cell RNA-Seq data from HEK293 cells. Consistent with expectation, the flux through known AS-NMD pathways is substantially higher than that captured by RNA-Seq. Our EJC RIPiT-Seq also definitively demonstrates that the splicing machinery itself has no ability to detect reading frame. We identified thousands of previously unannotated splicing events; while many can be attributed to “splicing noise”, others are evolutionarily-conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis. Conclusions: Deep sequencing of RNAs in post-splicing, pre-translational mRNPs provides a means to identify and quantify splicing events without the confounding influence of differential mRNA decay. For many known AS-NMD targets, the NMD-linked AS pathway predominates. EJC RIPiT-Seq also enabled identification of numerous conserved but previously unknown AS-NMD events.
2021-02-19 | GSE150228 | GEO
Project description:Nonsense-mediated mRNA decay (NMD) is a conserved mRNA quality control mechanism that identifies and destroys aberrant mRNAs that contain premature termination codons (PTCs). The NMD core comprises seven proteins, from SMG1 to SMG7. Arabidopsis has orthologues of most of these proteins. Studies on yeast, Drosophila, Humans and Arabidopsis reveal that NMD not only functions to target PTC-containing mRNAs but also acts as a global regulator of gene expression. It has also been reported that the NMD pathway branches with some target genes being dependent on specific NMD factors. In order to determine the extent to which different NMD factors co-regulate or independently regulate Arabidopsis genes, transcriptome analysis of smg7b-1 mutants will be carried out and added to existent data of upf1, upf3 and smg5 NMD mutants for comparison. RNA will be extracted from 17 day-old mutant and wild type seedlings grown at 22-24 C under constant light. 4 samples were used in this experiment
2011-10-06 | E-GEOD-32672 | biostudies-arrayexpress