“A memory of RPS25 loss drives resistance phenotypes”

In order to maintain cellular protein homeostasis, ribosomes are safeguarded against dysregulation by myriad processes. Remarkably, many cell types can withstand the genetic disruption of numerous ribosomal protein genes, indicating that select ribosome variants can sustain cellular life. Genetic alterations of ribosomal protein genes have been further linked to diverse cellular phenotypes and human disease, yet the direct and indirect consequences of sustained alterations in ribosomal protein levels are poorly understood. To address this knowledge gap, we studied and cellular consequences that follow genetic knockout of the small subunit ribosomal proteins RPS25 or RACK1 in a human haploid cell line. To our surprise, we found that multiple cellular phenotypes previously assumed to result from a direct effect on translation were instead caused by indirect effects. During characterization of ribosomes isolated from the RPS25 knockout cell line, we discovered a partial remodeling of the large subunit via the ribosomal protein paralog eL22L1. Upon further examination, we found that RPS25 knockout clones display irreversible rewiring of viral- and toxin-resistance phenotypes, suggesting that the cells had undergone a stable transition to a new cell state. This new state appears to drive pleiotropic phenotypes and is characterized by a dramatically altered transcriptome and membrane proteome. By homology-directed repair of a RPS25 knockout cell line, we demonstrate that even when RPS25 expression is rescued at the native genomic locus, cells fail to correct for the phenotypic hysteresis. Our results illustrate how the elasticity of cells to a ribosome perturbation can manifest as specific but indirect phenotypic outcomes.