Oral Presentation 28th Annual Lorne Proteomics Symposium 2023

Systematic analysis of post-translational modifications in the yeast ribosome reveals extensive heterogeneity (#63)

Joshua J Hamey 1 , Tara K Bartolec 1 , Marc R Wilkins 1
  1. Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia

Protein translation is intricately controlled within the eukaryotic cell. While previously thought to be controlled exclusively through translation factors, it has recently emerged that the ribosome itself is important for translational regulation. Ribosome composition is highly varied within cells, a phenomenon called ribosome heterogeneity, and this is known to regulate selective mRNA translation. However, the contribution of protein post-translational modifications (PTMs) to ribosome heterogeneity remains poorly understood. Here we have combined ribosome profiling through Ribo-Mega SEC (size exclusive chromatography) [1,2] with mass spectrometry to systematically profile ribosomal PTMs in Saccharomyces cerevisiae. Ribo-Mega SEC allowed separation of distinct pools of ribosomes, from translationally active polysomes to unincorporated subunits, for downstream mass spectrometric analysis. Through use of multiple different proteases for protein digestion, we identified every yeast ribosomal protein (RP) except one, with peptides covering every single residue for the vast majority of RPs. This includes paralogous pairs of RPs that differ by only a few residues. We successfully identified and quantified all 12 known methylation sites on RPs, confirming that these are all present on actively translating ribosomes. Remarkably, half of these methylation sites were found to be substoichiometric on polysomes, indicating that these methylation sites contribute to ribosome heterogeneity. We also identified 18 phosphorylation sites across 14 ribosomal proteins. These were found at low stoichiometry and on highly accessible, disordered regions of the ribosome, suggesting they may be actively regulated on intact ribosomes. Through open modification searches we identified several other PTMs on ribosomal proteins, including acetylation and ubiquitination. Lastly, quantification of paralogous RPs revealed that some pairs of paralogs are differentially incorporated into different populations of ribosomes. Together, our results reveal that PTMs contribute significantly to ribosome heterogeneity and provide a foundation for detailed studies on the roles of PTMs in translational regulation.

  1. [1] Yoshikawa, H., Larance, M., Harney, D. J., Sundaramoorthy, R., Ly, T., Owen-Hughes, T., & Lamond, A. I. (2018). Efficient analysis of mammalian polysomes in cells and tissues using Ribo Mega-SEC. Elife, 7.
  2. [2] Yoshikawa, H., Sundaramoorthy, R., Mariyappa, D., Jiang, H., & Lamond, A. I. (2021). Efficient and rapid analysis of Polysomes and ribosomal subunits in cells and tissues using Ribo mega-SEC. Bio-protocol, 11(15), e4106-e4106.