tRNA Synthetases Are Recruited to Yeast Ribosomes by rRNA Expansion Segment 7L but Do Not Require Association for Functionality

Protein biosynthesis is essential for any organism, yet how this process is regulated is not fully understood at the molecular level. During evolution, ribosomal RNA expanded in specific regions, referred to as rRNA expansion segments (ES). First functional roles of these expansions have only recently been discovered. Here we address the role of ES7La located in the large ribosomal subunit for factor recruitment to the yeast ribosome and the potential consequences for translation. Truncation of ES7La has only minor effects on ribosome biogenesis, translation efficiency and cell doubling. Using yeast rRNA deletion strains coupled with ribosome-specific mass spectrometry we analyzed the interactome of ribosomes lacking ES7La. Three aminoacyl-tRNA synthetases showed reduced ribosome association. Synthetase activities however remained unaltered suggesting that the pool of aminoacylated tRNAs is unaffected by the ES deletion. These results demonstrated that aminoacylation activities of tRNA synthetases per se do not rely on ribosome association. These findings suggest a role of ribosome-associated aminoacyl-tRNA synthetase beyond their core enzymatic functions.

Hypusinated eIF5A is required for the translation of collagen

Translation of mRNAs that encode peptide sequences with consecutive prolines (polyproline) requires the conserved and essential elongation factor eIF5A to facilitate the formation of peptide bonds. It has been shown that upon eIF5A depletion, yeast ribosomes stall in polyproline motifs, but also in tripeptide sequences that combine proline with glycine and charged amino acids. Mammalian collagens are enriched in putative eIF5A-dependent Pro-Gly-containing tripeptides. Here we show that depletion of active eIF5A in mouse fibroblasts reduced collagen 1 (Col1a1) content, which concentrated around the nuclei. Moreover, it provoked the up-regulation of endoplasmic reticulum (ER)-stress markers suggesting retention of partially synthesized Col1 in the ER. We confirmed that eIF5A is needed for heterologous collagen synthesis in yeast, and using a double luciferase reporter system we showed that eIF5A depletion interrupts translation at Pro-Gly-collagenic motifs. A dramatically lower level of Col1α1 protein was also observed in functional eIF5A-depleted human hepatic stellate cells treated with the profibrotic cytokine TGF-β1. In sum, our results show that collagen expression requires eIF5A and imply its potential as a target for regulating collagen production in fibrotic diseases.

Development, validation, and application of the ribosome separation and reconstitution system for protein translation in vitro

Stress-induced molecular damage to ribosomes can impact protein synthesis in cells, but cell-based assays do not provide a clear way to distinguish the effects of ribosome damage from stress responses and damage to other parts of the translation machinery. Here we describe a detailed protocol for the separation of yeast ribosomes from other translational machinery constituents, followed by reconstitution of the translation mixture in vitro. This technique, which we refer to as Ribosome Separation and Reconstitution (RSR), allows chemical modifications of yeast ribosomes without compromising other key translational components. In addition to the characterization of stress-induced ribosome damage, RSR can be applied to a broad range of experimental problems in studies of yeast translation.

Identification of changing ribosome protein compositions using cryo-EM and mass spectrometry

SummaryThe regulatory role of the ribosome in gene expression has come into sharper focus. It has been proposed that ribosomes are dynamic complexes capable of changing their protein composition in response to enviromental stimuli. We applied both cryo-EM and mass spectrometry to identify such changes in S. cerevisiae 80S ribosomes. Cryo-EM shows a fraction (17%) of the ribosome population in yeast growing in glucose lack the ribosomal proteins RPL10 (ul16) and RPS1A/B (eS1). Unexpectedly, this fraction rapidly increases to 34% after the yeast are switched to growth in glycerol. Using quantitative mass spectrometry, we found that the paralog yeast ribosomal proteins RPL8A (eL8A) and RPL8B (eL8B) change their relative proportions in the 80S ribosome when yeast are switched from growth in glucose to glycerol. Using yeast genetics and polysome profiling, we show that yeast ribosomes containing either RPL8A or RPL8B are not functionally interchangeable. Our combined cryo-EM and quantitative proteomic data support the hypothesis that ribosomes are dynamic complexes that alter their composition and functional activity in response to changes in growth or environmental conditions.

Functional divergence between the two P1–P2 stalk dimers on the ribosome in their interaction with ricin A chain

Wild-type yeast ribosomes and ribosomes bearing only the P1B–P2A dimer bound to the ricin A chain better and were more susceptible to depurination and toxicity than ribosomes bearing only the P1A–P2B dimer, indicating that the two stalk dimers differ in their function.