Ribosome Stoichiometry: From Form to Function

Abstract

The existence of eukaryotic ribosomes with distinct ribosomal protein (RP) stoichiometry and regulatory roles in protein synthesis has been speculated for over 60 years. Recent advances in mass spectrometry (MS) and high-throughput analysis have begun to identify and characterize distinct ribosome stoichiometry in yeast and mammalian systems. In addition to RP stoichiometry, ribosomes host a vast array of protein modifications, effectively expanding the number of human RPs from 80 to many thousands of distinct proteoforms. Is it possible that these proteoforms combine to function as a 'ribosome code' to tune protein synthesis? We outline the specific benefits that translational regulation by specialized ribosomes can offer and discuss the means and methodologies available to correlate and characterize RP stoichiometry with function. We highlight previous research with a focus on formulating hypotheses that can guide future experiments and crack the ribosome code.

Keywords: heterogeneity; mass spectrometry; ribosome; stoichiometry; translation.

Figure 1.. Timeline.

The concept of eukaryotic ribosome specialization has existed for decades, and recent methodological advances have resulted in renewed interest and the ability to explore and characterize these phenomena. In this timeline, a few key manuscripts are colored by the area of ribosome heterogeneity they have described.

Figure 2.. Heterogenous Ribosomes and their PTMs.

A) Ribosomes can be divided into the small (40S, shown in gold) and large (60S, shown in bronze) subunits, which in humans comprises 4 rRNAs (shown in blue) and 80 ribosomal proteins. B) Mass spectrometry analysis of human ribosomes reveals RPs are not all present at stoichiometric levels (Levels in monosomes compared to polysomes, unpublished data, U-937 human monocyte cells). C) RPs are highly modified with over 2500 modifications listed in Phosphositeplus [66] as of January 2018. The most abundant RP modifications currently known are phosphorylation, acetylation and methylation. Modification sites are shown in red. The human ribosome structures presented here were generated using PDB structure 5T2C [99] in the UCSF ChimeraX software.

Figure 3.. Ribosome specialization.

If populations of ribosomes exhibit distinct phenotypes there are multiple ways in which these functional differences could exist. A) Distinct ribosome subpopulations could have a range of specificities for their mRNAs. These could be from the individual mRNA level to global translation regulation. B) The timescale at which changes to RP stoichiometry or PTMs could exert effects on translation can potentially range from the extremely rapid/seconds (especially in the case of PTMs), to the very long term e.g. years. C) mRNA expression is noisy and buffered at the level of translation. D) The elongation rate of a ribosome represents a tradeoff between speed and accuracy. Further the elongation rate is not constant on a given mRNA with some sections of an mRNA being translated more rapidly than others.

Figure 4.. Identifying altered RP stoichiometry and PTMs.

A) For decades, the gold standard approach for isolating RPs in the context of intact, functional, ribosomes has been sucrose gradient centrifugation. B) Affinity purification is a powerful means of identifying differential RP association with complexes, however it cannot definitively say whether an RP is resident in a ribosome or represents an extra-ribosomal population of the RP. C) A combined approach whereby affinity purification is performed on sucrose gradient fractions allows the advantages of affinity purification to be applied to samples where the RP is known to be ribosome-resident. D) Heterogeneity amongst ribosomal protein modifications is a promising new area of research, and the methods required to explore this are an extension of those for identifying changes to RP association. Protease-digested peptides from sucrose gradient fractions or affinity purification can be enriched for a particular PTM of choice, either individually, or serially whereby the flow-through of one enrichment is applied to the next enrichment process.

Figure 5.. Testing for functional specialization.

A) To experimentally prove ribosome specialization, several outputs for measurement stand out. These are the mRNA binding specificity, elongation rates and error rates. A conclusive demonstration of functional ribosome specialization will likely employ several or all of these. B) If specific mRNAs are favored by individual ribosome conformations then this can be assessed by immunoprecipitation with tagged RPs. C) Elongation rates for the ribosome on particular mRNA substrates can be estimated from pulse-chase data. D) The error rate for individual ribosomes can be monitored using luminescent or fluorescent reporters for specific substrates, or in a higher-throughput manner by mass spectrometry. E) Functional validation of specialized ribosomes can be investigated through in vitro reconstitution of the phenotype.