Protein translation is the final step in gene expression and is carried out by the ribosome and dozens of translation-associated factors. Translation is therefore a critical control point for regulating cellular response to stress and environmental cues. However, how cellular signalling connects to and regulates the protein synthesis machinery remains poorly understood. Our recent work has focused on understanding how post-translational modifications of the translational machinery respond to stress and regulate translation in eukaryotes. In particular, we focus on investigating the roles that methylation and phosphorylation play in this process in both yeast and human cells.
Firstly, we systematically mapped modifications of yeast and human ribosomal proteins (RPs). To do this, we combined isolation of ribosomes using size exclusion chromatography with bottom-up mass spectrometry using different proteases. In combination with heavy methyl SILAC labelling and phosphopeptide enrichment, this revealed 12 methylation sites on yeast and human ribosomes, over 100 phosphorylation sites on yeast ribosomes and over 50 phosphorylation sites on human ribosomes.
To determine whether ribosomal phosphorylation is dynamically regulated in response to stress, we combined stable isotope labelling (SILAC) with ribosome isolation and phosphopeptide enrichment to measure changes in RP phosphorylation upon osmotic stress in yeast. Yeast cells were subject to 15 minutes of salt stress (0.7M NaCl) and polysomes, monosomes and ribosomal subunits were isolated. Digestion of ribosomal proteins with trypsin, LysC and GluC followed by phosphopeptide enrichment enabled the quantitative comparison of over 100 RP phosphosites between salt stress and unstressed yeast cells. Remarkably, we found that the vast majority of RP phosphosites were not regulated under these conditions, with a few notable exceptions. The most strongly regulated phosphosite was the highly conserved C-terminal phosphorylation of Rps6, which was significantly downregulated upon salt stress. This is in agreement with decades of research showing this phosphosite is strongly regulated upon stress across many eukaryotic species. Notably, we found a novel paralog-specific phosphorylation event that was increased approximately 4-fold upon osmotic stress. Given that paralog switching in response to stress has been observed previously, it is possible that phosphorylation mediates exchanges of paralogs upon stress, enabling reprogramming of translation. Going forward, we will investigate the effect of phospho-null (serine-to-alanine) mutations of these regulated phosphosites to measure their effects on cell growth, paralog incorporation, ribosomal population levels and mRNA selection under stress conditions.