Exploiting codon usage identifies RpS21 as anin vivosignal strength-dependent Ras/MAPK regulator

Abstract

Accumulating evidence suggests the evolutionarily conserved Ras/mitogen activated protein kinase (MAPK) signaling pathway directs distinct biological consequences that depend on distinct intensity states. Given this intensity-dependence, we hypothesized that different levels of Ras/MAPK signaling may be subjected to regulation by different sets of proteins. To identify such 'differential' signaling modifiers, we turned to the Drosophila eye as a model. First, we created flies whereby mutant active Ras V12 expressed in the eyes was enriched with either rare or commonly occurring codons. We show that this codon manipulation can generate either low or high levels of Ras protein and MAPK signaling, and correspondingly a mild or severe rough-eye phenotype. Using this novel platform to control Ras/MAPK signaling, we performed a wholegenome haploinsufficiency deficiency screen, which yielded 15 deficiencies that modify the rougheye phenotype in either high or low MAPK signaling states, but not both. We then mapped the underlying gene from one deficiency to the gene RpS21. Disrupting RpS21 expression increases MAPK signaling and enhances the rough-eye phenotype specifically when Ras V12 is encoded by rare codons (low signaling), and RpS21 negatively regulates Ras protein and MAPK signaling in several contexts. We also provide evidence that the MAPK pathway promotes expression of RpS21, providing potential negative feedback. Taken together, these data reveal intensityspecific regulation of Ras/MAPK signaling, with the first candidate of this class being RpS21.