Role of PML in repression of transposable elements and in embryonic stem cell fate
Ontology highlight
ABSTRACT:
INSTRUMENT(S): NextSeq 500
ORGANISM(S): Mus musculus
SUBMITTER: Valérie LALLEMAND-BREITENBACH
PROVIDER: E-MTAB-10153 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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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 established SMG8- and SMG9-depleted human osteosarcoma U2OS cells (Flp-In-T-REx-U2OS). 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 U2OS (WT or KO) cells were treated with DMSO.
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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 UPF1 gene is essential in cultured human cells and previous studies relied mostly on RNA interference to downregulate UPF1. Here we established an auxin-inducible UPF1 degron system in the human colorectal adenocarcinoma cell line HCT116 by first inserting the auxin receptor F-box protein-encoding AtAFB2-mCherry in the AAVS1 locus, followed by tagging UPF1 at the N-terminus or C-terminus with an V5-AID-tag (AID = miniIAA7 = AtIAA7 amino acids 37–104). With these cell lines we wanted to assess the transcriptome-wide expression changes upon rapid depletion of UPF1, estimate the effects of auxin treatment and compare N-terminal versus C-terminal tagging. To this end, depletion of UPF1 was induced with 500 µM indole-3-acetic acid (IAA) for various time periods (0-12h). As controls, the parental cell line (with AtAFB2-mCherry in the AAVS1 locus) or untreated cells were used.
2025-09-01 | E-MTAB-13839 | 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 UPF1 gene is essential in cultured human cells and previous studies relied mostly on RNA interference to downregulate UPF1. Here we established an auxin-inducible UPF1 degron system in the human colorectal adenocarcinoma cell line HCT116 by first inserting the auxin receptor F-box protein-encoding AtAFB2-mCherry in the AAVS1 locus, followed by tagging UPF1 at the N-terminus with an V5-AID-tag (AID = miniIAA7 = AtIAA7 amino acids 37–104). With this cell line we wanted to explore the time-resolved transcriptome-wide expression changes including the extent of NMD inhibition upon rapid depletion of UPF1. To this end, depletion of UPF1 was induced with 500 µM indole-3-acetic acid (IAA) for various time periods (0-48h). As controls, the parental cell line (with AtAFB2-mCherry in the AAVS1 locus) or untreated cells were used.
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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.
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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 UPF1 gene is essential in cultured human cells and previous studies relied mostly on RNA interference to downregulate UPF1. Here we established an dTAGV-1-inducible UPF1 degron system in the human colorectal adenocarcinoma cell line HCT116 or human embryonic kidney cell line HEK293 by tagging UPF1 at the N-terminus with an Myc-FKBP-tag (FKBP = FKBP12-F36V). With these cell lines we wanted to explore the transcriptome-wide expression changes including the extent of NMD inhibition upon rapid depletion of UPF1. To this end, depletion of UPF1 was induced with 0.25 µM dTAGV-1 for 12h. As controls, the parental cell line or untreated cells were used.
2025-09-01 | E-MTAB-13829 | 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 UPF1 gene is essential in cultured human cells and previous studies relied mostly on RNA interference to downregulate UPF1. Here we established an auxin-inducible UPF1 degron system in the human colorectal adenocarcinoma cell line HCT116 by first inserting the auxin receptor F-box protein-encoding AtAFB2-mCherry in the AAVS1 locus, followed by tagging UPF1 at the N-terminus with an V5-AID-tag (AID = miniIAA7 = AtIAA7 amino acids 37–104). Using SLAM-Seq and this cell line, we wanted to explore the time-resolved RNA stability changes upon rapid depletion of UPF1. To this end, depletion of UPF1 was induced with 500 µM indole-3-acetic acid (IAA) for various time periods (0h, 12h and 24h) and the cells were labeled with 200 µM 4-thiouridine (4SU) the last 2 hours before harvesting. As controls, the parental cell line (with AtAFB2-mCherry in the AAVS1 locus) or unlabeled cells were used.
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