biostudies-arrayexpressVolker BöhmHomo sapiensSTAR read aligner (version 2.7.10b)https://www.ebi.ac.uk/biostudies/studies/E-MTAB-13829UPF1 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.biostudies-arrayexpressSequencing - 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.Library Construction - Libraries were prepared from 500ng total RNA with the Illumina® Stranded mRNA Preparation kit. ERCC RNA Spike-In Mix (Thermo Fischer) was added to the samples before library preparation. After poly-A selection (using Oligo(dT) magnetic beads), mRNA was purified, fragmented and reverse transcribed with random hexamer primers. Second strand synthesis with dUTPs was followed by A-tailing, adapter ligation and library amplification (12 cycles) to create the final cDNA libraries. After library validation and quantification (Agilent Tape Station), equimolar amounts of library were pooled.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.Sample Treatment - 5x10^5 cells were seeded in 6-well plates one day before starting the depletion experiment. UPF1 depletion was induced with 0.25 µM dTAGV-1 (Tocris Bioscience; Cat# 6914) for 12 hours. All cells were harvested at the same time to minimize differences in cell numbers.Growth Protocol - Both control and FKBP-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). For control and FKBP-UPF1-tagged HEK293 cell lines, DMEM with high glucose and GlutaMAX supplement (Gibco; Cat# 61965059) was used and supplemented as described above.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.Niels GehringVolker BöhmfalseRNA-Seq of UPF1 depletion in human colorectal adenocarcinoma cell line HCT116 or human embryonic kidney cell line HEK293 via the dTAG degron 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 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.2025-09-01T00:00:00Z2025-09-26T17:40:39.488Z2024-03-04T12:28:25.567ZE-MTAB-1382940934927ERP157628E-MTAB-13836E-MTAB-13837E-MTAB-13839E-MTAB-13788E-MTAB-13789E-MTAB-14725E-MTAB-14755E-MTAB-13787EFO_0002944EFO_0004170EFO_0003789EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_000396910.1016/j.molcel.2025.08.015