Variable stoichiometry among core ribosomal proteins

1. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not. Understanding the regulation and structure of the eukaryotic ribosome is essential to understanding protein synthesis and its deregulation in disease. While ribosomes are believed to have a fixed stoichiometry among their core ribo-somal proteins (RPs), some experiments suggest a more variable composition. Reconciling these views requires direct and precise quantification of RPs. We used mass-spectrometry to directly quantify RPs across monosomes and polysomes of budding yeast and mouse embryonic stem cells (ESC). Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function. Introduction In eukaryotes, the most–studied molecular mechanisms for regulating protein translation are mediated by eukaryotic initiation factors that activate mRNAs for translation by the ribosomes and can be modulated by microRNAs and RNA binding proteins (1–5). Eukaryotic ribosomes are believed to have a fixed composition of 4 ribosomal RNAs and 80 core RPs (5–8), some of which are represented by several paralogous RPs. However, studies of eukaryotic ribosomes (9–16) have demonstrated that (i) genetic perturbations to the core RPs specifically affect the translation of some mRNAs and not others and (ii) mRNAs coding for core RPs are transcribed, spliced, and translated differentially across physiological conditions (17–23). These results suggest the hypothesis (24–26) that, depending on the tissue type and the physiological conditions, cells can alter the stoichiometry among the core RPs comprising the ribosomes and thus in 2. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not. turn alter the translational efficiency of distinct mRNAs. However, differential RP–expression can reflect extra ribosomal functions of the RPs (9, 27, 28). Furthermore, polysomes (multiple ribosomes per mRNA) from different cell–lines have similar core RP stoichiometries (29). Thus, the variable RP–stoichiometry in the ribosomes of wild-type cells that is suggested by the ribosome specialization hypothesis remains unproven. Results Variable stoichiometry among core RPs in yeast To measure whether the stoichiometry among RPs can change …