RNA-Seq of UPF1, SMG5 or SMG6 depletion in human embryonic kidney cell line HEK293 via the dTAG degron system and siRNA-mediated knockdowns
Nonsense-mediated decay (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. UPF1 is the central NMD factor required for PTC recognition and subsequent recruitment of the executing factors SMG5 and SMG6. To study the impact of UPF1, SMG5 or SMG6 protein depletion on the transcriptome, we established dTAGV-1-inducible degron systems in the human embryonic kidney cell line HEK293 (Flp-In-T-REx-293) by tagging the respective protein at the N-terminus with an Myc-FKBP-tag (FKBP = FKBP12-F36V). FKBP-tagged SMG5 and SMG6 cell lines were treated for 72h with 60 pmol siRNAs targeting the respective mRNA, whereas FKBP-tagged UPF1 and the parental cell line was treated similarly with control Luciferase siRNA. Protein degradation of UPF1, SMG5 and SMG6 was induced with 0.25 µM dTAGV-1 for 24h (48h after RNAi), whereas control cells were treated with DMSO.