RNA-seq of Bafilomycin-treated cells

Nonsense-mediated decay (NMD) is a pathway that degrades messenger RNAs containing premature termination codons. Here, a genome-wide screen for NMD factors uncovered an unexpected mechanism that broadly governs 3ʹ untranslated region (UTR)-directed regulation. The screen revealed that NMD requires lysosomal acidification, which allows transferrin-mediated iron uptake, which in turn is necessary for iron-sulfur (Fe-S) cluster biogenesis. This pathway converges on the Fe-S cluster-containing ribosome recycling factor ABCE1, whose impaired function results in the movement of ribosomes into 3ʹ UTRs where they displace exon junction complexes, thereby abrogating NMD. Importantly, these effects extend beyond NMD substrates, with ABCE1 activity required to maintain the accessibility of 3ʹ UTRs to diverse regulators, including microRNAs and RNA binding proteins. Due to the sensitivity of the Fe-S cluster of ABCE1 to iron availability and reactive oxygen species, these findings reveal an unanticipated vulnerability of 3ʹ UTR-directed regulation to lysosomal dysfunction, iron deficiency, and oxidative stress. Method: RNA-seq was performed in HCT116 NMD reporter cells that were either treated with DMSO or 5 nM bafilomycin for 48 hours. Two replicates in each condition, and thus 4 biological samples were used for these experiments.

无义介导的mRNA降解（nonsense-mediated decay, NMD）是一类可降解携带提前终止密码子的信使RNA（messenger RNA, mRNA）的细胞通路。本研究通过全基因组筛选NMD相关因子，发现了一种可广泛调控3'非翻译区（3' untranslated region, UTR）介导基因表达调控的意外机制。筛选结果表明，NMD通路依赖于溶酶体酸化：该过程可支持转铁蛋白介导的铁摄取，而铁摄取又是铁硫簇（iron-sulfur, Fe-S）生物合成的必要前提。该通路的核心作用靶点为含有Fe-S簇的核糖体回收因子ABCE1；当其功能受损时，核糖体会迁移至3' UTR区域并驱逐该处的外显子连接复合物，从而阻断NMD通路。值得注意的是，上述效应并不局限于NMD底物，ABCE1的活性对于维持3' UTR对多种调控因子（包括微小RNA（microRNA, miRNA）和RNA结合蛋白（RNA binding protein, RBP））的可及性至关重要。鉴于ABCE1的Fe-S簇对铁可用性及活性氧（reactive oxygen species, ROS）具有敏感性，本研究揭示了3' UTR介导的基因调控对溶酶体功能障碍、铁缺乏及氧化应激存在此前未被认知的易感性。实验方法：将HCT116 NMD报告细胞系分别用二甲基亚砜（dimethyl sulfoxide, DMSO）或5 nM巴弗洛霉素处理48小时后进行RNA测序（RNA-sequencing, RNA-seq）；每个处理组设置两个生物学重复，本实验共使用4个生物学样本。