SLAM-Seq of UPF1 depletion in human colorectal adenocarcinoma cell line HCT116 via the auxin-inducible degron (AID) system
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 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). Using SLAM-Seq and this cell line, we wanted to explore the time-resolved RNA stability changes upon rapid depletion of UPF1. To this end, depletion of UPF1 was induced with 500 µM indole-3-acetic acid (IAA) for various time periods (0h, 12h and 24h) and the cells were labeled with 200 µM 4-thiouridine (4SU) the last 2 hours before harvesting. As controls, the parental cell line (with AtAFB2-mCherry in the AAVS1 locus) or unlabeled cells were used.