biostudies-arrayexpressVolker BöhmHomo sapiensSTAR read aligner (version 2.7.10b)https://www.ebi.ac.uk/biostudies/studies/E-MTAB-13837UPF1 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 lines we performed Ribo-Seq (ribosome footprinting) to assess the effects of UPF1 depletion on translation. To this end, depletion of UPF1 was induced with 500 µM indole-3-acetic acid (IAA) for 12h. As control untreated cells were used.biostudies-arrayexpressSample Collection - The cells were washed with PBS containing 100 μg ml−1 cycloheximide (Biochemika) before cell lysis. Samples were lysed in polysome buffer (100 mM Tris-HCl pH 7.4, 750 mM NaCl, 25 mM MgCl2, 1 mM dithiothreitol (DTT), 1% Triton X-100, 100 μg ml−1 cycloheximide and 25 U ml−1 Turbo DNase (Thermo Fisher Scientific)) and directly frozen in liquid nitrogen.Library Construction - 5000 ng of the isolated monosomes were depleted of rRNA using a RiboPool kit for Ribosome Profiling (siTOOLs Biotech). The RNA was separated on a 17% Urea PAA gel, 27–30 nt RNA fragments were eluted from the gel and 5’ extremity phosphorylated with 10 U T4 polynucleotide kinase (New England Biolabs) for 1 h at 37 °C. For library generation, the NextFlex small RNA sequencing kit (PerkinElmer) was used according to the manufacturer’s instructions.Growth Protocol - 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 (Gibco), supplemented with 10% fetal bovine serum (Gibco) and 2 mM L-glutamine (Gibco).Sample Treatment - 3x10^6 cells were seeded a 10 cm dish one day before starting the depletion experiment. UPF1 depletion was induced with 500 µM indole-3-acetic acid (IAA; Sigma-Aldrich; Cat# I5148) for 12 hours. All cells were harvested at the same time to minimize differences in cell numbers.Nucleic Acid Extraction - RNA was then digested with 2400 U ml−1 RNase I (Ambion) for 45 min in slow agitation at RT and digestion stopped with 640 U ml−1 SUPERase•In (Thermo Fisher Scientific). Monosomes were purified by size exclusion with Illustra MicroSpin S-400 HR columns (GE Healthcare) and extracted with 3 volumes of Trizol LS (Thermo Fisher Scientific), chloroform and the RNA Clean & Concentrator kit (Zymo- Research).Sequencing - The pool was quantified by using the KAPA Library Quantification Kit and sequenced on an Illumina NovaSeq6000 sequencer with 2×100bp protocol.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Demultiplexed, adapter-trimmed FASTQ files for individual samples were merged and only read 1 was used for the analysis as follows: UMIs (random 4 bases at the 5′ and 3′ ends) were extracted with UMI-tools (version 1.1.2; https://github.com/CGATOxford/UMI-tools), length-filtered (≥ 16 & ≤ 40 nts) and mapped with Bowtie2 (version 2.5.0) against human rRNA, tRNA, miRNA and snoRNA. Unmapped 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). Deduplication was performed using the extracted UMIs with UMI-tools.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.Ribo-seqHomo 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 BöhmfalseRibo-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). With this cell lines we performed Ribo-Seq (ribosome footprinting) to assess the effects of UPF1 depletion on translation. To this end, depletion of UPF1 was induced with 500 µM indole-3-acetic acid (IAA) for 12h. As control untreated cells were used.2025-09-01T00:00:00Z2025-09-25T13:04:32.268Z2024-02-22T10:08:45.747ZE-MTAB-1383740934927ERP164400E-MTAB-13836E-MTAB-13839E-MTAB-13788E-MTAB-13789E-MTAB-14725E-MTAB-14755E-MTAB-13787E-MTAB-13829EFO_0002944EFO_0004170EFO_0003789EFO_0008891EFO_0005518EFO_0003816EFO_0004184EFO_000396910.1016/j.molcel.2025.08.015