ZNF574 is a Quality Control Factor for Defective Ribosome Biogenesis Intermediates [CRISPRi_FLOW]

Eukaryotic ribosome assembly is an intricate process that involves four ribosomal RNAs, 80 ribosomal proteins, and over 200 biogenesis factors that take part in numerous interdependent steps. The complexity and essentiality of this process creates opportunities for deleterious mutations to occur, accumulate, and impact downstream cellular processes. “Dead-end” ribosome intermediates that result from biogenesis errors are rapidly degraded, affirming the existence of quality control pathway(s) that monitor ribosome assembly. However, the factors that differentiate between on-path and dead-end intermediates are unknown. We engineered a system to perturb ribosome assembly in human cells and discovered that faulty ribosomes are degraded via the ubiquitin proteasome system. We identified ZNF574 as a key component of a novel quality control pathway, which we term the Ribosome Assembly Surveillance Pathway (RASP). In an animal model, loss of ZNF574 leads to developmental defects, further emphasizing the importance of RASP in organismal health. Overall design: To identify quality control factors targeting defective large subunits, we generated K562 dCas9-KRAB cells that stably express RFP-tagged mutant uL16. The CRISPRi compact library (hCRISPRi_dual_1_2 pooled library, Addgene, Cat#187246) was transduced in duplicate into K562 CRISPRi cells stably expressing uL16mut-RFP at an MOI < 1 (percentage of transduced cells 2 days after transduction: 20%–40%). Replicates were maintained separately in four T125 flasks per replicate for the course of the screen. Two days after transduction, the cells were selected with 1 mg/mL puromycin for 3 days, at which point transduced cells accounted for 80%–95% of the population. Cells were allowed to recover to >80% cell viability, as measured with a Cell Countess (Invitrogen). The cells were maintained in T125 flasks by daily dilution to 0.5 × 10^6 cells/mL at an average coverage of greater than 1000 cells per sgRNA for the duration of the screen. Cells were sorted using BD FACS Aria3 two days after recovery, based on the RFP fluorescence of the uL16mut-RFP reporter. Cells with the highest (~30%) and lowest (~30%) RFP expression were collected and snap-frozen. Approximately 10 million cells were collected per bin.

真核生物核糖体组装是一项精密复杂的过程，涉及4种核糖体RNA（ribosomal RNA）、80种核糖体蛋白以及超过200种生物发生因子，参与众多相互依赖的步骤。该过程的复杂性与必要性使得有害突变得以产生、积累，并影响下游细胞过程。由生物发生错误产生的“停滞型”核糖体中间体可被快速降解，这证实了监测核糖体组装的质量控制通路的存在。然而，区分正常组装通路中间体与停滞型中间体的因子仍未明确。我们构建了可在人类细胞中扰动核糖体组装的体系，发现缺陷核糖体可通过泛素-蛋白酶体系统（ubiquitin proteasome system）降解。我们鉴定出ZNF574作为新型质量控制通路的关键组分，将该通路命名为核糖体组装监视通路（Ribosome Assembly Surveillance Pathway）。在动物模型中，ZNF574的缺失会导致发育缺陷，进一步凸显了RASP在生物体健康中的重要性。

整体实验设计：为鉴定靶向缺陷型核糖体大亚基的质量控制因子，我们构建了稳定表达RFP标记的突变uL16的K562 dCas9-KRAB细胞。将CRISPRi紧凑型文库（hCRISPRi_dual_1_2 pooled library，Addgene，货号Cat#187246）以多重感染复数<1的条件，以双重复样本形式转导至稳定表达uL16mut-RFP的K562 CRISPRi细胞中（转导后2天的转导细胞占比为20%~40%）。每个重复组分别接种于4个T125培养瓶中完成整个筛选过程。转导后2天，用1 mg/mL嘌呤霉素筛选3天，此时转导细胞占细胞群体的80%~95%。待细胞恢复至存活率>80%（采用Invitrogen公司Cell Countess细胞计数仪检测）后，将细胞维持于T125培养瓶中，每日稀释至0.5×10^6 cells/mL，整个筛选期间每个sgRNA的平均覆盖细胞数大于1000个。恢复培养2天后，采用BD FACS Aria3流式细胞仪，基于uL16mut-RFP报告基因的RFP荧光信号对细胞进行分选。收集RFP表达量最高（约30%）与最低（约30%）的细胞并快速冷冻。每个分选组分收集约1000万个细胞。