RNA-sequencing in SH-SY5Y neuroblastoma cell line with TDP-43 KD and nuclear and cytoplasmic fractionation as well and treatment with the nonsense-mediated decay inhibition using SMG1-11j inhibitor
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 2500, NA
ORGANISM(S): Homo sapiens
SUBMITTER: Anna-Leigh Brown
PROVIDER: E-MTAB-15653 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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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:Nonsense-mediated mRNA decay (NMD) is a translation-dependent mRNA turnover pathway, which degrades transcripts containing premature termination codons. The execution of NMD requires the phosphorylation of N- and C-terminal tails of the key NMD factor UPF1, which thereby serve as binding platforms for the degradation factors SMG5, SMG6 and SMG7. UPF1 phosphorylation is mediated by the kinase SMG1, whose activity is regulated by a heterodimer consisting of SMG8 and SMG9. Recent work indicated that SMG9 functions as a bridge between SMG1 and SMG8, allowing the C-terminus of SMG8 to elicits its role of stabilizing the autoinhibitory state of SMG1. Here, we deleted the C-terminus of endogenous SMG8 in human colorectal adenocarcinoma cell line HCT116 via CRISPR-Cas9. In addition, we established SMG8- and SMG9-depleted cells. With these cell lines we wanted to explore the regulatory role of SMG8 and SMG9 for NMD execution. Furthermore, we tested the transcriptomic changes upon treatment of cells with the SMG1 inhibitor SMG1i, which functions as an ATP-competitive inhibitor and binds to the active site of SMG1. Cells were treated with 0, 0.1 or 1 μM SMG1i for 24 h. As controls, the HCT116 wildtype cells were treated with DMSO.
2026-02-13 | E-MTAB-13949 | biostudies-arrayexpress
Project description:UPF1 is a multi-domain RNA helicase that constantly monitors the transcriptome by non-specifically binding to mRNAs, dissociating from non-target transcripts, and initiating degradation on selected target RNAs via multiple proposed pathways such as nonsense-mediated decay (NMD). NMD is a translation-coupled mechanism that targets mRNAs harboring a premature stop codon (PTC) for degradation, thereby serving as a quality control and gene regulatory pathway ensuring transcriptome integrity. The execution of NMD requires the phosphorylation of N- and C-terminal tails of the key NMD factor UPF1, which thereby serve as binding platforms for the degradation factors SMG5, SMG6 and SMG7. UPF1 phosphorylation is mediated by the kinase SMG1, which catalytic activity can be inhibited with the SMG1 inhibitor SMG1i, a small molecule that functions as an ATP-competitive inhibitor and binds to the active site of SMG1. We wanted to assess the transcriptome-wide expression changes upon inhibition of SMG1. To this end, we treated human foreskin fibroblast (HFF) and human umbilical vein endothelial cells (HUVEC) with 1 µM SMG1i inhibitor for 24h. As controls, cells were treated with DMSO for 24h.
2025-09-01 | E-MTAB-14755 | biostudies-arrayexpress
Project description:SMARD1 is an infantile autosomal recessive motor neuron (MN) disease, caused by mutations in the Immunoglobulin mu binding protein 2 (IGHMBP2). We investigated the potential of a spinal cord neural stem cell population isolated on the basis of aldehyde dehydrogenase activity (ALDH) to modify disease progression of nmd mice, an animal model of SMARD1. ALDHhiSSClo stem cells are self-renewing and multipotent and when intratechally transplanted in nmd mice generate motor neurons properly localized in the spinal cord ventral horns. Transplanted nmd animals presented delayed disease progression, sparing of motor neurons and ventral root axons and increased life-span. To further investigate the molecular events responsible for these differences, microarray and Real time RT-PCR analysis of wild-type, mutated and transplanted nmd spinal cord were undertaken. We demonstrated a down-regulation of genes involved in excitatory amino acid toxicity and oxidative stress handling, as well as an up-regulation of genes related to the chromatin organization in nmd compared to wild-type mice, suggesting that they may play a role in SMARD1 pathogenesis. Spinal cord of nmd transplanted mice expressed high transcript levels for genes related to neurogenesis such as Doublecortin (DCX), LIS1 and drebrin. The presence of DCX-expressing cells in adult nmd spinal cord suggests that both exogenous and endogenous neurogenesis may contribute to the observed nmd phenotype amelioration. Experiment Overall Design: We intratechally transplanted a spinal cord neural stem cell population isolated on the basis of aldehyde dehydrogenase activity (ALDH) in nmd mice, an animal model of SMARD1. Experiment Overall Design: We analyzed the lumbar spinal cord tract of transplanted nmd mice (n=3), untransplanted nmd mice (n=3) and wild-type mice (n=3) (all mice are of male gender). The latter groups underwent surgical procedure with vehicle. The surgical procedure and cell transplantation were done at P1 and the animals were sacrificed at 6 weeks of age.
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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:UPF3A and UPF3B are paralogous genes in human cells that are involved in the nonsense-mediated decay (NMD) pathway. NMD is a cellular quality control mechanism that monitors mRNAs during translation. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction activates NMD and leads to the degradation of the mRNA. To investigate the role of UPF3B and UPF3A in NMD, we have generated UPF3B knockout (KO) and UPF3A-UPF3B double KO (dKO) human Flp-In T-REx 293 cells using CRISPR-Cas9. We generated RNA-Sequencing data for wildtype, UPF3B KO and UPF3A-UPF3B dKO cells with additional siRNA-mediated knockdown of Luciferase (Luc) as control or UPF3B.
2022-04-25 | E-MTAB-10716 | biostudies-arrayexpress
Project description:The functional coupling between alternative pre-mRNA splicing (AS) and the mRNA quality control mechanism called nonsense-mediated decay (NMD) has been shown to modulate the expression of numerous genes. Previous studies have identified several examples of this regulation in developing neurons. However, the systems-level effects of AS-NMD in this context have been poorly understood. To this end, we performed a longitudinal analysis of gene expression in mouse embryonic stem cells undergoing induced neuronal differentiation, as well as primary neural cells. To improve the detection of NMD-sensitive splice forms, some of the samples were treated with the translation inhibitor cycloheximide or separated into nuclear and cytoplasmic fractions. Our analyses uncovered hundreds of AS-NMD events with significant potential to regulate their host genes. Notably, these events were significantly overrepresented in developmentally downregulated genes. Particularly strong enrichment was detected for alternative cassette exons that trigger NMD upon their inclusion into mature mRNA. Overall, this study extends our understanding of the molecular mechanisms underlying neuronal differentiation and highlights the importance of post-transcriptional regulation gene expression in orchestrating this intricate process.
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Project description:UPF3A and UPF3B are paralogous genes in human cells that are involved in the nonsense-mediated decay (NMD) pathway. NMD is a cellular quality control mechanism that monitors mRNAs during translation. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction activates NMD and leads to the degradation of the mRNA. To investigate the role of UPF3B and UPF3A in NMD, we have generated UPF3A overexpressing human HeLa Flp-In T-REx cells using the PiggyBac transposon system. We generated RNA-Sequencing data for wild type and UPF3A overexpressing (OE) cells.
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2015-06-29 | PXD001668 | Pride
Project description:Transactive response DNA binding protein 43 kDa (TDP-43) is a protein that regulates splicing, the loss of which underlies the pathophysiology of a variety of neurodegenerative disorders, including amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). In disease states, TDP-43 is cleared from the nucleus of neurons, leading to the loss of its splicing repressor function and the aberrant inclusion of cryptic exons. Frequent inclusion of premature termination codons (PTCs) by cryptic exons leads to degradation of cryptic mRNA by nonsense-mediated mRNA decay (NMD). However, detection of cryptic exons relies on inefficient NMD; therefore cryptic exons efficiently targeted and eliminated by NMD remain undetectable through conventional RNA-sequencing. Here we generated a comprehensive set of neuronal targets of TDP-43 in human IPSC-derived i3Neurons (i3N) by combining TDP-43 knockdown with inhibition of several factors regulating NMD, and identified various cryptic exons that were either underestimated or entirely undetected by TDP-43 knockdown alone.
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