Project description:Monosome and disome profiling was performed on Flag-STAU1 Flp-In 293 T-REx to study the causes of ribosomal collisions, and whether this may be modulated by the presence/absence of Staufen-1. Cells were treated with either an siRNA targeting STAU1 transcript (4x samples) or a control siRNA (2x samples). Two of the four samples treated with the STAU1 siRNA had siRNA-resistant STAU1 mRNA expression induced by doxycycline (rescue). Sequencing libraries from monosome and disome fractions were generated in parallel from the same samples. Note that unique molecular identifiers/random barcodes (UMIs/RBCs) were included in the sequencing experiment. Each UMI has been moved to the fastq read name of each read. For example \\"xxxxxxrbc:AGCCAAT\\" in the read name signifies that the given read had a UMI of \\"AGCCAAT\\". Using these UMIs, PCR duplicates can be removed with UMI-Tools following read alignment.
Project description:UPF3A and UPF3B are paralogous genes in human cells that are involved in the nonsense-mediated decay (NMD) pathway. NMD is a cellular quality control mechanism that monitors mRNAs during translation. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction activates NMD and leads to the degradation of the mRNA. To investigate the role of UPF3B and UPF3A in NMD, we have generated UPF3B knockout human Flp-In T-REx 293 cells using CRISPR-Cas9. We generated RNA-Sequencing data for wildtype and UPF3B KO cells with additional siRNA-mediated knockdown of Luciferase (Luc) as control or UPF3A.
Project description:RNPS1 is a splicing regulatory protein and a component of the ASAP/PSAP complex, which is associated with the exon junction complex and modulates alternative splicing. It was previously postulated that the isolated RRM domain of RNPS1 in complex with ASAP/PSAP is able to regulate certain alternative splicing events. We aimed to investigate in Flp-In T-REx 293 cells which alternative splicing events are rescued by the expression of the wild type RNPS1 or the isolated RRM domain of RNPS1 in a RNPS1 knockdown background by using RNA-Seq analyses. The rescue construct was stably integrated into the genome using the PiggyBac transposon system. As controls, either Luciferase (Luc) siRNA was used or RNPS1 was knocked down without rescue.
Project description:UPF3A and UPF3B are paralogous genes in human cells that are involved in the nonsense-mediated decay (NMD) pathway. NMD is a cellular quality control mechanism that monitors mRNAs during translation. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction activates NMD and leads to the degradation of the mRNA. To investigate the role of UPF3B and UPF3A in NMD, we have generated UPF3B knockout (KO) and UPF3A-UPF3B double KO (dKO) human Flp-In T-REx 293 cells using CRISPR-Cas9. We generated RNA-Sequencing data for wildtype, UPF3B KO and UPF3A-UPF3B dKO cells with additional siRNA-mediated knockdown of Luciferase (Luc) as control or UPF3B.
Project description:UPF3A and UPF3B are paralogous genes in human cells that are involved in the nonsense-mediated decay (NMD) pathway. NMD is a cellular quality control mechanism that monitors mRNAs during translation. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction activates NMD and leads to the degradation of the mRNA. To investigate the role of UPF3B and UPF3A in NMD, we have generated UPF3A overexpressing human HeLa Flp-In T-REx cells using the PiggyBac transposon system. We generated RNA-Sequencing data for wild type and UPF3A overexpressing (OE) cells.
Project description:UPF3A and UPF3B are paralogous genes in human cells that are involved in the nonsense-mediated decay (NMD) pathway. NMD is a cellular quality control mechanism that monitors mRNAs during translation. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction activates NMD and leads to the degradation of the mRNA. To investigate the role of UPF3B and UPF3A in NMD, we have generated UPF3A knockout (KO) human Flp-In T-REx 293 cells using CRISPR-Cas9, as well as FLAG-tagged UPF3A overexpressing cells using the PiggyBac transposon system. We generated RNA-Sequencing data for wildtype, UPF3A KO and UPF3A overexpressing (OE) cells, in part with additional siRNA-mediated knockdown of Luciferase (Luc) as control or UPF3B.
Project description:Expression profile of human Flp-In-293-WT-FBXO25 or Flp-In-293-∆F-FBXO25 cells comparing non treated cells vs. tetraciclyne treated for 24 and 48 hours. Flp-In-293-∆F-FBXO25 cells when induced express the non functional protein (without F-box domain). The objective of the study was to identify genes up or down-regulated when Fbxo25 wild-type or Fbxo25 lacking F-box domain is superexpressed.