Loss of a Conserved tRNA Anticodon Modification Perturbs Cellular Signaling

Abstract

Transfer RNA (tRNA) modifications enhance the efficiency, specificity and fidelity of translation in all organisms. The anticodon modification mcm ⁵s ²U ³⁴ is required for normal growth and stress resistance in yeast; mutants lacking this modification have numerous phenotypes. Mutations in the homologous human genes are linked to neurological disease. The yeast phenotypes can be ameliorated by overexpression of specific tRNAs, suggesting that the modifications are necessary for efficient translation of specific codons. We determined the in vivo ribosome distributions at single codon resolution in yeast strains lacking mcm ⁵s ²U. We found accumulations at AAA, CAA, and GAA codons, suggesting that translation is slow when these codons are in the ribosomal A site, but these changes appeared too small to affect protein output. Instead, we observed activation of the GCN4-mediated stress response by a non-canonical pathway. Thus, loss of mcm ⁵s ²U causes global effects on gene expression due to perturbation of cellular signaling.

Author Summary

Ribosomes translate the messages of the genetic code into functional proteins with the help of transfer RNAs (tRNAs), which carry a specific amino acid at one end and recognize three letters of the genetic code (a codon) with the other. tRNAs are subject to extensive chemical modifications, which are thought to enhance the efficiency, fidelity and specificity of translation. Many of these modifications are conserved across all domains of life, underscoring their biological importance. Despite intensive biochemical characterization, the physiological roles of most tRNA modifications are unknown. The tRNA modification mcm ⁵s ²U ³⁴ is required for normal growth and stress resistance in yeast; mutants lacking this modification have numerous phenotypes. Mutations in the homologous human genes are linked to neurological disease. The yeast phenotypes can be ameliorated by overexpression of specific tRNAs, suggesting that the modifications are necessary for efficient translation of specific codons. In order to test this model, we determined ribosome distributions at each codon in yeast strains lacking mcm ⁵s ²U. The changes we found appeared too small to affect protein output. Instead, we observed a non-canonical activation of a yeast stress response pathway. Thus, loss of mcm ⁵s ²U causes widespread perturbation of cellular signaling, independent of any codon-specific translation defects.