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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-Seq of WT Flp-In T-REx 293 and UPF3B knockout cell lines with additional siRNA-mediated control or UPF3A knockdowns

ABSTRACT: 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 human Flp-In T-REx 293 cells using CRISPR-Cas9. We generated RNA-Sequencing data for wildtype and UPF3B KO cells with additional siRNA-mediated knockdown of Luciferase (Luc) as control or UPF3A.

INSTRUMENT(S): Illumina HiSeq 4000

ORGANISM(S): Homo sapiens

SUBMITTER: Volker Böhm

PROVIDER: E-MTAB-10711 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Human UPF3A and UPF3B enable fault-tolerant activation of nonsense-mediated mRNA decay.

Wallmeroth Damaris D Lackmann Jan-Wilm JW Kueckelmann Sabrina S Altmüller Janine J Dieterich Christoph C Boehm Volker V Gehring Niels H NH

The EMBO journal 20220422 10

The paralogous human proteins UPF3A and UPF3B are involved in recognizing mRNAs targeted by nonsense-mediated mRNA decay (NMD). UPF3B has been demonstrated to support NMD, presumably by bridging an exon junction complex (EJC) to the NMD factor UPF2. The role of UPF3A has been described either as a weak NMD activator or an NMD inhibitor. Here, we present a comprehensive functional analysis of UPF3A and UPF3B in human cells using combinatory experimental approaches. Overexpression or knockout of U ...[more]

PMID: 35451084

Similar Datasets

RNA-Seq of WT Flp-In T-REx 293, UPF3B knockout and UPF3A-UPF3B double knockout cell lines with additional siRNA-mediated control or UPF3B knockdowns

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

RNA-Seq of WT Flp-In T-REx 293, UPF3A knockout and UPF3A overexpression cell lines in part with additional siRNA-mediated control or UPF3B knockdowns

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 knockout (KO) human Flp-In T-REx 293 cells using CRISPR-Cas9, as well as FLAG-tagged UPF3A overexpressing cells using the PiggyBac transposon system. We generated RNA-Sequencing data for wildtype, UPF3A KO and UPF3A overexpressing (OE) cells, in part with additional siRNA-mediated knockdown of Luciferase (Luc) as control or UPF3B.

2022-08-31 | E-MTAB-10718 | biostudies-arrayexpress

RNA-Seq of WT HeLa Flp-In T-REx and UPF3A overexpression cell lines

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.

2022-08-31 | E-MTAB-11184 | biostudies-arrayexpress

Proximity labelling of FLAG-TurboID-tagged UPF3B wild type and interaction-deficient mutant proteins followed by proteomics analysis

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 in NMD, we have generated FLAG-TurboID-UPF3B wild type or mutant expressing human Flp-In T-Rex 293 UPF3B knockout cells using the PiggyBac transposon system. We generated proteomic data from the proximity labeled interactome of these UPF3B variants.

2022-05-04 | PXD029898 | Pride

Proximity labelling of FLAG-TurboID-tagged UPF3B wild type and interaction-deficient mutant proteins followed by proteomics analysis

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 in NMD, we have generated FLAG-TurboID-UPF3B wild type or mutant expressing human Flp-In T-Rex 293 UPF3B knockout cells using the PiggyBac transposon system. We generated proteomic data from the proximity labeled interactome of these UPF3B variants.

2022-05-04 | PXD032337 | Pride

Deciphering the cellular roles and dissecting functional regions of UPF3A and UPF3B in human NMD

Project description:The paralogous proteins UPF3A and UPF3B are involved in recognizing defective mRNAs that are degraded by nonsense-mediated mRNA decay (NMD). While UPF3B has been demonstrated to support NMD, UPF3A was reported to act either an NMD activator or an NMD inhibitor. Here, we present a comprehensive functional analysis of UPF3A and UPF3B in human cells using overexpression, knockdowns, knockouts (KO) and rescue experiments. Overexpression or knockout of UPF3A did not result in detectable changes in global NMD activity or other specific transcriptome alterations . NMD activity was also virtually unchanged in UPF3B KO cells. In contrast, the co-depletion or co-knockout of UPF3A and UPF3B resulted in a marked NMD inhibition and a global upregulation of PTC-containing transcripts. In rescue experiments UPF3B was fully functional when either UPF2- or EJC binding was impaired . However, the deletion of both interaction sites or one interaction site and a central region of UPF3B impaired its NMD function. Altogether, our work identifies critical functional domains of UPF3B and establishes redundant roles of UPF3A and UPF3B during NMD.

2022-05-04 | PXD027120 | Pride

Genome-wide consequences of compromised NMD and their relavence for variable clinical phenotype of patients with UPF3B mutations [mRNA profiling]

2011-11-01 | GSE27125 | GEO

Genome-wide consequences of compromised NMD and their relavence for variable clinical phenotype of patients with UPF3B mutations [RNA-seq]

2011-11-01 | GSE27199 | GEO

Genome-wide consequences of compromised NMD and their relavence for variable clinical phenotype of patients with UPF3B mutations [CNV]

2011-11-01 | GSE27412 | GEO

Genome-wide consequences of compromised NMD and their relavence for variable clinical phenotype of patients with UPF3B mutations [CNV]

Project description:Nonsense-mediated mRNA decay (NMD) surveillance pathways are best known to be involved in the degradation of mRNA with premature termination codons (PTCs). More recent studies demonstrate that the role of NMD pathways goes well beyond the degradation of PTC containing mRNA, into the regulation of cell function and thus normal development. We have taken advantage of the availability of naturally occurring loss of function mutations in the UPF3B gene, a major component of the exon junction complex (EJC), to inquire about genome-wide consequences of compromised NMD. We identify that about 5% of the lymphoblastoid cell transcriptome is directly or indirectly impacted upon in patients with UPF3B mutations with minimal effect on alternative splicing. We identify UPF3A-NMD as a likely, major modifier of the UPF3B patient phenotype through variable UPF3A protein stabilisation. Among the most consistently deregulated direct targets of UPF3B-NMD we identify the ARHGAP24 as the most likely gene implicated in the neuronal phenotype of UPF3B patients. To assess the impact of UPF3B-NMD deficiency on human transcriptome, we sequenced polyA RNA extracted from lymphoblastoid cell lines of patients (n=4) and controls (n=2). We complemented the analysis using Affymetrix Human Exon 1.0 St array using total RNA of the same cell line from patients (n=5, 3 of whom were also sequenced) and controls (n=5). Moreover, we overlapped identified differently expressed genes with copy number variation data of the patients, obtained using Illumina Human Omniexpress chip, to exclude possible false positive.

2011-11-01 | E-GEOD-27412 | biostudies-arrayexpress

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