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. 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.