RNA-seq using mouse Neuro-2A (N2A) neuroblastoma cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE180136 | GEO | 2021/10/25
REPOSITORIES: GEO
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Project description:To identify high-confidence NMD targets in mouse N2A neuroblastoma cells, we used our established transcriptome-wide RNA sequencing (RNA-seq) methodologies. Through parallel analyses of RNA-seq upon UPF1-knockdown (KD) and RNA immunoprecipitation (RIP-seq) footprinting of p-UPF1-bound RNAs, we identified 1027 high-confidence neuronal NMD targets.
2021-10-25 | GSE180134 | 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.
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:Nonsense-mediated mRNA decay (NMD) is a conserved RNA surveillance pathway that is an important modulator of disease pathology and is required for embryonic development. Despite significant research effort, the rules that govern NMD remain incompletely understood. Here we used a combined¬ approach, integrating RNA-Seq, ribosome footprinting, and CLIP-Seq analysis of the essential NMD factor Upf1, to provide a more complete picture of the role of NMD in modulating gene expression in murine embryonic stem cells (mESCs). We show that presence of an exon-exon junction ?50 nucleotides (nt) downstream of a termination codon (dEJ) contributes to NMD independently of 3' UTR length, but has stronger effects in genes with shorter 3' UTRs. We also map translated upstream open reading frames (uORFs) in mESCs and show that they are associated with NMD regulation, especially of genes encoding transcription factors, and we find that lowly translated mRNAs can escape NMD. Finally, we identify over 200 direct binding targets of Upf1 and describe a pathway of Upf1-dependent gene regulation reliant on Upf1 binding to the 3' UTR and independent of presence of a dEJ. Together, these analyses characterize known and discover novel determinants of NMD and establish a broader role in mESC gene regulation for Upf1. mRNA-Seq analysis of wildtype (2 samples), translationally inhibited (by cycloheximide treatment, 2 samples), control-depleted (2 samples), and Upf1-depleted (4 samples) mouse embryonic stem cells (mESCs); CLIP-Seq analysis of Upf1 (5 samples, and 5 samples of IgG control CLIP-Seq); Ribosome footprint profiling of wildtype (1 sample), control-depleted (1 sample), and Upf1-depleted (1 sample) mESCs
2013-06-12 | E-GEOD-41785 | biostudies-arrayexpress
Project description:Nonsense-mediated mRNA decay (NMD) is a conserved RNA surveillance pathway that is an important modulator of disease pathology and is required for embryonic development. Despite significant research effort, the rules that govern NMD remain incompletely understood. Here we used a combined¬ approach, integrating RNA-Seq, ribosome footprinting, and CLIP-Seq analysis of the essential NMD factor Upf1, to provide a more complete picture of the role of NMD in modulating gene expression in murine embryonic stem cells (mESCs). We show that presence of an exon-exon junction ≥50 nucleotides (nt) downstream of a termination codon (dEJ) contributes to NMD independently of 3' UTR length, but has stronger effects in genes with shorter 3' UTRs. We also map translated upstream open reading frames (uORFs) in mESCs and show that they are associated with NMD regulation, especially of genes encoding transcription factors, and we find that lowly translated mRNAs can escape NMD. Finally, we identify over 200 direct binding targets of Upf1 and describe a pathway of Upf1-dependent gene regulation reliant on Upf1 binding to the 3' UTR and independent of presence of a dEJ. Together, these analyses characterize known and discover novel determinants of NMD and establish a broader role in mESC gene regulation for Upf1.
2013-06-12 | GSE41785 | GEO
Project description:To investigate selectivity of mRNA oxidation, total RNA and oxidized RNA isolated from Neuro 2a cells before and after H2O2 treatment were employed for microarray analysis. It was found that selective oxidation of mRNA already occurs under normal culture conditions but was increased by H2O2, especially in a subset of mRNAs related to certain functions. Moreover, mRNA oxidation level is also related to its abundance or stability (half-life time). This shows for the first time that mRNA oxidation is associated with RNA homeostasis including function, stability and abundance depending on cellular redox status in a genome-wide scale. Neuro 2a cells received hydrogen peroxide treatment or no treatment as a control. Samples were applied for RNA extraction and ARP labeling, which could bind with apurinic/apyrimidinic sites, and then a pull-down process to isolate oxidized RNA. Total RNA and oxidized RNA were used for subsequent transcriptomic profiling. 4 types of samples were analyzed: Basal-total: untreated N2a cells labeled with ARP, but not processed for the pull-down assay. Ox-total: hydrogen peroxide-treated N2a cells labeled with ARP, but not processed for the pull-down assay. Basal-ARP: untreated N2a cells labeled with ARP, and processed for the pull-down assay. ARP-derivatized RNA, which is also oxidized RNA, was concentrated and used for the microarray analysis. Ox-ARP: hydrogen peroxide-treated N2a cells labeled with ARP, and processed for the pull-down assay. ARP-derivatized RNA, which is also oxidized RNA, was concentrated and used for the microarray analysis.
2012-10-01 | E-GEOD-39641 | biostudies-arrayexpress
Project description:The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited “closed” state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active “open” state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower nucleic acid binding kinetics and enhanced ATP-stimulated nucleic acid dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2, and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.
2024-05-29 | GSE246484 | GEO
Project description:The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited “closed” state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active “open” state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower nucleic acid binding kinetics and enhanced ATP-stimulated nucleic acid dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2, and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.
2024-05-29 | GSE246483 | GEO
Project description:Cells were grown to ~50% confluency before RNA polymerase activity was blocked with 10 µg/mL Actinomycin D (Sigma) in DMSO. Control cells were treated with DMSO alone. Transcriptional inhibition of N2A cells was conducted for 32 h with cells harvested at time zero (0 h) and after 30 min, 2, 4, 8, 16 and 32 h. Inhibition of 3T3 cells was conducted for 24 h with harvesting at time zero (0 h) and after 30 min, 2, 4, 8 and 24 h.
2012-01-24 | E-MTAB-961 | biostudies-arrayexpress
Project description:Nonsense-mediated mRNA decay (NMD) is a major translation-dependent RNA degradation pathway required for embryo development and telomere maintenance. Core NMD factors Upf1, Upf2 and Upf3 are conserved from yeast to mammals but a model of the NMD machinery compatible with all eukaryotes is not yet available. We performed the first large-scale quantitative characterization of yeast NMD complexes through affinity purification and mass-spectrometry with 7 different NMD-related factors, with or without Rnase, in strains deleted or not for NMD genes. This extensive characterization of NMD complexes identified two distinct complexes associated with Upf1: Detector (Upf1/2/3) and Effector. Effector contained, in addition to Upf1, the mRNA decapping enzyme and two potential equivalents of mammalian Smg6/5/7: Nmd4 and Ebs1. Like the Smg proteins, Nmd4 and Ebs1 were required for efficient NMD. Our results suggest that the core eukaryotic NMD machinery is conserved across species and operates through successive Upf1-bound Detector and Effector complexes.
2018-03-12 | PXD007159 | Pride