biostudies-arrayexpressVolker BöhmHomo sapiensSTAR read aligner (version 2.7.10b)https://www.ebi.ac.uk/biostudies/studies/E-MTAB-13836UPF1 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.biostudies-arrayexpressGrowth 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 - 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 12 and 24 hours before harvesting). Except unlabeled N-AID-UPF1 controls, all cells were labeled with 200 µM 4-thiouridine (4SU; Abcam; Cat# ab143718) for 2 hours before harvesting. All cells were harvested at the same time to minimize differences in cell numbers.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. Thiol modification was performed as previously described by Herzog et al. 2017. Shortly, we mixed 1 µg of RNA with 10 mM Iodoacetamide (IAA; Sigma-Aldrich; Cat#I6125), 50 mM NaPO4 (pH 8) and 50 % DMSO. The reaction was performed at 50°C for 15 minutes, protected from light. Alkylation was quenched with 20mM DTT. RNA was precipitated by adding 2.5 volumes of 100% EtOH and 1 µg GlycoBlue (Ambion; Cat# AM9515) and incubating -80°C for 30 minutes. After cold centrifugation at 16,000 x g for 30 minutes, the pellet was washed with 75% EtOH, re-centrifuged for 10 minutes, air-dried for 5-10 minutes, and resuspended in 5-10 µl of H2O.Library Construction - poly(A) mRNA isolation and library preparation was carried out with 500 ng RNA as input using the NEBNext Poly (A) mRNA magnetic isolation module (NEB; Cat#E7490L) and NEBNext Ultra II Directional RNA library preparation kit (NEB; Cat#E7760L) according to the manufacturer's instructions.Sequencing - The pool was quantified by using the Peqlab KAPA Library Quantification Kit and sequenced on an Illumina NovaSeq6000 sequencer with 2×100bp protocol, targeting approximately 20 million reads.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).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData 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).MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000The 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.RNA-seq of coding RNAHomo sapiensRapid UPF1 depletion illuminates the temporal dynamics of the NMD-regulated human transcriptome.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.Markus LandthalerVolker BöhmfalseSLAM-Seq of UPF1 depletion in human colorectal adenocarcinoma cell line HCT116 via the auxin-inducible degron (AID) systemUPF1 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.2025-09-01T00:00:00Z2025-09-25T15:07:44.504Z2024-02-22T10:03:52.025ZE-MTAB-1383640934927ERP157649E-MTAB-13837E-MTAB-13839E-MTAB-13788E-MTAB-13789E-MTAB-14725E-MTAB-14755E-MTAB-13787E-MTAB-13829EFO_0002944EFO_0004170EFO_0003789EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_000396910.1016/j.molcel.2025.08.015