{"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-13949"],"description":["Nonsense-mediated mRNA decay (NMD) is a translation-dependent mRNA turnover pathway, which degrades transcripts containing premature termination codons. 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, whose activity is regulated by a heterodimer consisting of SMG8 and SMG9. Recent work indicated that SMG9 functions as a bridge between SMG1 and SMG8, allowing the C-terminus of SMG8 to elicits its role of stabilizing the autoinhibitory state of SMG1. Here, we deleted the C-terminus of endogenous SMG8 in human colorectal adenocarcinoma cell line HCT116 via CRISPR-Cas9. In addition, we established SMG8- and SMG9-depleted cells. With these cell lines we wanted to explore the regulatory role of SMG8 and SMG9 for NMD execution. Furthermore, we tested the transcriptomic changes upon treatment of cells with the SMG1 inhibitor SMG1i, which functions as an ATP-competitive inhibitor and binds to the active site of SMG1. Cells were treated with 0, 0.1 or 1 μM SMG1i for 24 h. As controls, the HCT116 wildtype cells were treated with DMSO."],"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.","Growth Protocol - HCT-116 (human, male, colorectal carcinoma, epithelial; ATCC, cat. no. CCL-247; RRID:CVCL_0291) and Flp-In-T-REx-293 (human, female, embryonic kidney, epithelial; Thermo Fisher Scientific, cat. no. R78007; RRID:CVCL_U427) were cultivated in high glucose, GlutaMAX DMEM (Gibco; Cat# 61965-026) supplemented with 9% fetal bovine serum (Gibco; Cat# 10270106) and 1x Penicillin-Streptomycin (Gibco; Cat# 15140122). The cells were cultured at 37 °C and 5% CO2 in a humidified incubator.","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 - 3x10^5 cells were seeded in 6-well plates. After 24h, different final concentrations (0, 0.1 or 1 μM) of SMG1 inhibitor (SMG1i; DOI: 10.1016/j.bmcl.2012.08.107) dissolved in DMSO were added. After 24 h cells were harvested.","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 50 million clusters per sample.","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"],"study_type":["RNA-seq of coding RNA"],"species":["Homo sapiens"],"pubmed_authors":["Niels Gehring","Volker Böhm"],"additional_accession":[]},"is_claimable":false,"name":"RNA-Seq of SMG1-SMG8-SMG9 complex alterations in human colorectal adenocarcinoma cell line HCT116","description":"Nonsense-mediated mRNA decay (NMD) is a translation-dependent mRNA turnover pathway, which degrades transcripts containing premature termination codons. 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, whose activity is regulated by a heterodimer consisting of SMG8 and SMG9. Recent work indicated that SMG9 functions as a bridge between SMG1 and SMG8, allowing the C-terminus of SMG8 to elicits its role of stabilizing the autoinhibitory state of SMG1. Here, we deleted the C-terminus of endogenous SMG8 in human colorectal adenocarcinoma cell line HCT116 via CRISPR-Cas9. In addition, we established SMG8- and SMG9-depleted cells. With these cell lines we wanted to explore the regulatory role of SMG8 and SMG9 for NMD execution. Furthermore, we tested the transcriptomic changes upon treatment of cells with the SMG1 inhibitor SMG1i, which functions as an ATP-competitive inhibitor and binds to the active site of SMG1. Cells were treated with 0, 0.1 or 1 μM SMG1i for 24 h. As controls, the HCT116 wildtype cells were treated with DMSO.","dates":{"release":"2026-02-13T00:00:00Z","modification":"2026-02-13T02:02:13.503Z","creation":"2024-04-12T17:12:02.438Z"},"accession":"E-MTAB-13949","cross_references":{"ENA":["ERP159404"],"EFO":["EFO_0002944","EFO_0004170","EFO_0003789","EFO_0005518","EFO_0003816","EFO_0003738","EFO_0004184","EFO_0003969"]}}