RiboMinus RNA-Seq of UPF1 depletion in human colorectal adenocarcinoma cell line HCT116 via the auxin-inducible degron (AID) system. RiboMinus RNA-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). With this experiment using rRNA depletion during library preparation, we wanted to explore the time-resolved transcriptome-wide expression changes, especially of non-poly(A) RNAs, upon rapid depletion of UPF1. To this end, depletion of UPF1 was induced with 500 µM indole-3-acetic acid (IAA) for two time periods (12h or 48h). As control, the parental cell line (with AtAFB2-mCherry in the AAVS1 locus) was used.

UPF1是一种多结构域RNA解旋酶，可通过非特异性结合mRNA、从非靶标转录本上解离，并通过无义介导的mRNA降解（nonsense-mediated decay, NMD）等多种已提出的通路对选定的靶标RNA启动降解，以此持续监控细胞转录组。无义介导的mRNA降解是一种与翻译偶联的分子机制，可靶向携带提前终止密码子（premature stop codon, PTC）的mRNA进行降解，因此作为质量控制与基因调控通路，保障转录组的完整性。UPF1基因在体外培养的人类细胞中是必需基因，既往相关研究大多依靠RNA干扰（RNA interference, RNAi）技术下调UPF1的表达水平。本研究在人类结直肠腺癌细胞系HCT116中构建了生长素诱导型UPF1降解标签系统：首先将编码生长素受体F-box蛋白的AtAFB2-mCherry插入AAVS1基因位点，随后在UPF1的N端融合添加V5-AID标签（AID = miniIAA7 = AtIAA7氨基酸残基37~104）。本实验在文库制备阶段采用了核糖体RNA（ribosomal RNA, rRNA）去除策略，旨在探究UPF1快速降解后，全转录组范围内的时间依赖性表达变化，尤其是非多聚腺苷酸化RNA（non-poly(A) RNAs）的表达差异。为此，我们使用500 μM吲哚-3-乙酸（indole-3-acetic acid, IAA）诱导UPF1降解，设置了两个处理时长（12小时或48小时）；对照组则使用仅在AAVS1位点整合了AtAFB2-mCherry的亲本细胞系。