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

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 wild-type or mutant uL16. We transduced these cells with a whole-genome CRISPRi library, selected infected cells by treating them with puromycin for three days, and then maintained the cells at 0.5 × 10^6 cells/ml with viability over 90% for ten cell doublings. After ten cell doublings, we harvested the cells, isolated genomic DNA, and constructed a sequencing library.