biostudies-arrayexpressVolker BöhmHomo sapiensSTAR read aligner (version 2.7.10b)https://www.ebi.ac.uk/biostudies/studies/E-MTAB-14755UPF1 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 execution of NMD requires the phosphorylation of N- and C-terminal tails of the key NMD factor UPF1, which thereby serve as binding platforms for the degradation factors SMG5, SMG6 and SMG7. UPF1 phosphorylation is mediated by the kinase SMG1, which catalytic activity can be inhibited with the SMG1 inhibitor SMG1i, a small molecule that functions as an ATP-competitive inhibitor and binds to the active site of SMG1. We wanted to assess the transcriptome-wide expression changes upon inhibition of SMG1. To this end, we treated human foreskin fibroblast (HFF) and human umbilical vein endothelial cells (HUVEC) with 1 µM SMG1i inhibitor for 24h. As controls, cells were treated with DMSO for 24h.biostudies-arrayexpressSample Treatment - 5x10^5 cells were seeded in 6-well plates one day before starting the SMG1i treatment. SMG1 inhibitor (SMG1i; DOI: 10.1016/j.bmcl.2012.08.107) dissolved in DMSO was added to a final concentration of 1 µM and cells were harvested 24 hours later.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 - HFF (TERT-HFF; Ximbio Cat# 154459) and HUVEC (HUVEC/TERT 2; ATCC Cat# CRL-4053; RRID:CVCL_9Q53) cell lines were maintained at 37°C and 5% CO2 in a humidified incubator in (HFF:) DMEM + 10% FBS, + 1% MEM Sigma, Cat# M7145-100ml) or (HUVEC:) EGM Endothelial Cell Growth Medium BulletKit (Lonza, Cat# CC-3124) supplemented with 10% FBS and 20 µg/ml G418.Sequencing - The pool was quantified by using the KAPA Library Quantification Kit and sequenced on an Illumina NovaSeq6000 sequencer with 150+150bp protocol, in two batches targeting approximately 20 million reads.Sample Collection - Cells were harvested and lysed by adding 500 µl Invitrogen™ TRIzol™ Reagent (Invitrogen; Cat# 15596018) to each well.Library Construction - poly(A) mRNA isolation and library preparation was carried out with 600 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.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 BöhmfalseRNA-Seq of SMG1 inhibition via SMG1i in human foreskin fibroblast (HFF) and human umbilical vein endothelial cells (HUVEC)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 execution of NMD requires the phosphorylation of N- and C-terminal tails of the key NMD factor UPF1, which thereby serve as binding platforms for the degradation factors SMG5, SMG6 and SMG7. UPF1 phosphorylation is mediated by the kinase SMG1, which catalytic activity can be inhibited with the SMG1 inhibitor SMG1i, a small molecule that functions as an ATP-competitive inhibitor and binds to the active site of SMG1. We wanted to assess the transcriptome-wide expression changes upon inhibition of SMG1. To this end, we treated human foreskin fibroblast (HFF) and human umbilical vein endothelial cells (HUVEC) with 1 µM SMG1i inhibitor for 24h. As controls, cells were treated with DMSO for 24h.2025-09-01T00:00:00Z2025-09-25T15:27:45.091Z2025-01-20T21:24:13.256ZE-MTAB-1475540934927ERP168491E-MTAB-13836E-MTAB-13837E-MTAB-13839E-MTAB-13788E-MTAB-13789E-MTAB-14725E-MTAB-13787E-MTAB-13829EFO_0002944EFO_0004170EFO_0003789EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_000396910.1016/j.molcel.2025.08.015