Abstract
In order to maintain cellular protein homeostasis, ribosomes are safeguarded against dysregulation by myriad processes. Remarkably, many cell types can withstand genetic lesions of certain ribosomal protein genes, some of which are linked to diverse cellular phenotypes and human disease. Yet the direct and indirect consequences from these lesions are poorly understood. To address this knowledge gap, we studied in vitro and cellular consequences that follow genetic knockout of the ribosomal proteins RPS25 or RACK1 in a human cell line, as both proteins are implicated in direct translational control. Prompted by the unexpected detection of an off-target ribosome alteration in the RPS25 knockout, we closely interrogated cellular phenotypes. We found that multiple RPS25 knockout clones display viral- and toxin-resistance phenotypes that cannot be rescued by functional cDNA expression, suggesting that RPS25 loss elicits a cell state transition. We characterized this state and found that it underlies pleiotropic phenotypes and has a common rewiring of gene expression. Rescuing RPS25 expression by genomic locus repair failed to correct for the phenotypic and expression hysteresis. Our findings illustrate how the elasticity of cells to a ribosome perturbation can drive specific phenotypic outcomes that are indirectly linked to translation and suggests caution in the interpretation of ribosomal protein gene mutation data.
Highlights
The eukaryotic ribosome is comprised of four strands of ribosomal RNA (rRNA) and ∼80 ribosomal proteins (RPs), most of which are essential for life
Since no clear differences in C9orf72 66-repeat (C9-66R) mRNA stability were observed in the previous study [22], these findings suggest that posttranslational mechanisms of dipeptide clearance may instead explain the strong reduction in polyGA in the HAP1 RPS25 knockout
Our results illustrate that cellular adaptation to ribosomal protein loss, rather than direct translation control, can drive phenotypes assumed to result from preferential translation
Summary
The eukaryotic ribosome is comprised of four strands of rRNA and ∼80 ribosomal proteins (RPs), most of which are essential for life. To ensure accurate and efficient protein synthesis, cells have evolved numerous measures to control and protect the cellular ribosome pool. The existence of genetic knockouts of select RPs in yeast and human cell lines indicates that cells are elastic to ribosome compositional alterations [1,2]. The presence of ribosomes with substoichiometric RP levels in unperturbed cells has raised the possibility that certain alterations might represent direct, regulated control of protein synthesis by RPs [3,4]. Alterations could represent ribosomes that have escaped from imperfect cellular quality control measures. RP loss may drive both direct effects on translation and indirect effects as cells sense and adapt to ribosome irregularities. While genetic RP loss is linked to numerous cellular phenotypes and human disease, the mechanistic basis by which these alterations arise remains unclear [5]
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