Ribosome profiling of ABCE1-knockout 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 3' 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 3' 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 3' 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 3' UTR-directed regulation to lysosomal dysfunction, iron deficiency, and oxidative stress. Overall design: Method: Ribosome profiling was performed in HCT116 cells that received, via lentivirus, Cas9 and sgRNAs targeting either ABCE1 or non-targeting control sgRNAs. Two sgRNAs were used for 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，发现了一种可广泛介导3'非翻译区（3' untranslated region, UTR）靶向调控的意外机制。筛选结果表明，NMD依赖溶酶体酸化过程——该过程可介导转铁蛋白依赖的铁摄取，而铁摄取正是铁硫簇（iron-sulfur cluster, Fe-S）生物合成的必要前提。该通路最终汇聚于含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区域并置换外显子连接复合物，进而阻断NMD过程。值得注意的是，此类效应并不局限于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）及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）的敏感性，本研究揭示了3' UTR靶向调控对溶酶体功能障碍、铁缺乏及氧化应激存在未被预见的易感性。整体实验设计：实验方法：本研究以HCT116细胞为模型，通过慢病毒载体向细胞内递送靶向ABCE1的Cas9与sgRNA，以及非靶向对照sgRNA，随后开展核糖体谱分析（ribosome profiling）。每个实验组使用2条sgRNA，因此本实验共设置4份生物学样本。