{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Volker Böhm"],"organism":["Homo sapiens"],"software":["STAR read aligner (version 2.7.10b)"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-13788"],"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."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Library Construction - Libraries were prepared using the Illumina Stranded TruSeq RNA sample preparation kit. ERCC RNA Spike-In Mix (Thermo Fischer) was added to the samples before library preparation. Library preparation started with 500ng total RNA. After poly-A selection (using poly-T oligo-attached magnetic beads), mRNA was purified and fragmented using divalent cations under elevated temperature. The RNA fragments underwent reverse transcription using random primers. This was followed by second strand cDNA synthesis with DNA Polymerase I and RNase H. After end repair and A-tailing, indexing adapters were ligated. The products were then purified and amplified (15 cycles) to create the final cDNA libraries. After library validation and quantification (Agilent Tape Station), equimolar amounts of library were pooled.","Sequencing - The pool was quantified by using the Peqlab KAPA Library Quantification Kit and the Applied Biosystems 7900HT Sequence Detection System. The pool was sequenced on an Illumina NovaSeq6000 sequencing instrument with a PE100 protocol aiming for 75 million clusters per sample.","Nucleic Acid Extraction - Total RNA was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research; Cat# R2052) including the recommended DNase I treatment according to the manufacturer's instructions.","Growth Protocol - Both control and N-AID-UPF1-tagged HCT116 cell lines were maintained at 37°C and 5% CO2 in a humidified incubator in McCoy's 5A (Modified) Medium with GlutaMAX supplement (Gibco; Cat# 36600088), supplemented with 9% fetal bovine serum (Gibco; Cat# 10270106) and 1x Penicillin-Streptomycin (Gibco; Cat# 15140122).","Sample Treatment - 2.5x10^5 cells were seeded in 6-well plates one day before starting the depletion experiment. UPF1 depletion was induced with 500 µM indole-3-acetic acid (IAA; Sigma-Aldrich; Cat# I5148) at various time points during the experiment (between 0 and 48 hours before harvesting). For the 48-hour time point, the medium was exchanged once for fresh medium with IAA. All cells were harvested at the same time to minimize differences in cell numbers.","Sample Collection - Cells were harvested and lysed by adding 1 ml of in-house prepared TRI reagent to each well (prepared following DOI: 10.1371/journal.pbio.3000107)."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - Reads were aligned against the human genome (GRCh38, GENCODE release 42 transcript annotations supplemented with SIRVomeERCCome annotations from Lexogen; obtained from https://www.lexogen.com/sirvs/download/) using the STAR read aligner (version 2.7.10b, https://github.com/alexdobin/STAR)."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq 6000"],"pubmed_abstract":["The RNA helicase UPF1 shapes the transcriptome as the core factor of nonsense-mediated mRNA decay (NMD). The essential role of UPF1 in human cells has impeded efforts to delineate its directly regulated transcripts and molecular function. To investigate the effects of rapid UPF1 depletion, we engineered human cell lines with endogenous UPF1 fused to conditional degron tags. Temporal-resolution transcriptomic analyses identified direct target mRNAs, consisting predominantly of NMD substrates that are mostly stabilized within hours of UPF1 depletion. By integrating long-read sequencing and ribosome profiling data, we defined the consolidated NMD-regulated human transcriptome (NMDRHT), uncovering previously unannotated transcripts and establishing alternative splicing as a major contributor of NMD-targeted mRNAs. Additionally, we identified non-canonical NMD events that lack indication of being driven by other UPF1-dependent degradation routes. Our work refines the role of the post-transcriptional regulator UPF1 and introduces an experimentally validated NMD-regulated transcriptome as a navigable resource at https://nmdrht.uni-koeln.de."],"study_type":["RNA-seq of coding RNA"],"species":["Homo sapiens"],"pubmed_title":["Rapid UPF1 depletion illuminates the temporal dynamics of the NMD-regulated human transcriptome."],"pubmed_authors":["Boehm V, Wallmeroth D, Wulf PO, Popp O, Teixeira Alves LG, Reinecke L, Riedel M, Wyler E, Franitza M, Becker K, Polkovnychenko K, Del Giudice S, Benlasfer N, Mertins P, Landthaler M, Gehring NH.","Niels Gehring","Volker Böhm"],"additional_accession":[]},"is_claimable":false,"name":"RNA-Seq of UPF1 depletion in human colorectal adenocarcinoma cell line HCT116 via the auxin-inducible degron (AID) system","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.","dates":{"release":"2025-09-01T00:00:00Z","modification":"2025-09-30T14:08:06.021Z","creation":"2024-02-20T19:33:40.758Z"},"accession":"E-MTAB-13788","cross_references":{"pubmed":["40934927"],"ENA":["ERP157495"],"Biostudies":["E-MTAB-13836","E-MTAB-13837","E-MTAB-13839","E-MTAB-13789","E-MTAB-14725","E-MTAB-14755","E-MTAB-13787","E-MTAB-13829"],"EFO":["EFO_0002944","EFO_0004170","EFO_0003789","EFO_0005518","EFO_0003816","EFO_0003738","EFO_0004184","EFO_0003969"],"doi":["10.1016/j.molcel.2025.08.015"]}}