Ribosome heterogeneity and specialisation

Somatic ribosomal protein defects such as missense mutations in RPL10 (uL16) and RPS15 (uS19) as well as somatic copy number losses of RPL5 (uL18), RPL11 (uL5) and RPL22 (eL22) have been described in a variety of hematologic and solid malignancies. We previously characterised the T-cell leukemia associated RPL10-R98S mutation using a genomewide translatome analysis (proteome, polysomal RNA-seq, Ribo-seq and total mRNA-seq). This revealed that RPL10-R98S generates a specialised ribosome that hypertranslates oncoproteins as JAK-STAT signaling components, anti-apoptotic protein BCL2 and serine/glycine synthesis enzyme PSPH. To verify whether other cancer-associated somatic ribosome defects rewire translation in a similar manner, we generated an onco-ribosome cell line library by CRISPR-Cas9 containing isogenic lymphoid cell clones (wild type, Rpl5+/-, Rpl11+/-, Rpl22+/-, Rpl22-/-, Rpl10-R98S, Rps15-P131S and Rps15-H137Y). Genomewide translatome analysis of this library reveals little translational changes in the RP knock-out cell lines. By contrast, the analysed RP point mutations are associated with profound translational rewiring, which is most pronounced for the Rps15 mutants. A significant impact of these Rps15 mutations on the ribosomal structure and translation dynamics is further supported by cryo-EM analyses. The genes presenting significant alteration of translation efficiency in Rpl10 and Rps15 point mutants show an enrichment for transcriptional regulators, revealing extensive cross-talk between translational and transcriptional regulation in RP point mutant cells. Furthermore, Dr De Keersmaecker's results support that loss of function mutations in Rpl5, Rpl11 and Rpl22 impose little translational rewiring, suggesting that these genotypes rather result in extra-ribosomal pro-oncogenic effects.

Translational regulation impacts both pluripotency maintenance and cell differentiation. To what degree the ribosome exerts control over this process remains unanswered. Accumulating evidence has demonstrated heterogeneity in ribosome composition in various organisms. 2'-O-methylation (2'-O-me) of rRNA represents an important source of heterogeneity, where site-specific alteration of methylation levels can modulate translation. Here, Professor Lund examines changes in rRNA 2'-O-me during mouse brain development and tri-lineage differentiation of human embryonic stem cells (hESCs). He found distinct alterations between brain regions, as well as clear dynamics during cortex development and germ layer differentiation. Professor Lund identifies a methylation site impacting neuronal differentiation. Modulation of its methylation levels affects ribosome association of the fragile X mental retardation protein (FMRP) and is accompanied by an altered translation of WNT pathway-related mRNAs. Together, these data identify ribosome heterogeneity through rRNA 2'-O-me during early development and differentiation and suggest a direct role for ribosomes in regulating translation during cell fate acquisition.

Dr Thompson's group has previously shown that eS25 is required for efficient translation by non-canonical mechanisms of initiation. In order to understand the role of eS25 in cellular homeostasis they performed a proteomics analysis in primary human renal proximal epithelial cells. These studies revealed that cell cycle proteins were decreased upon eS25 knockdown. Indeed, a cell cycle analysis showed that eS25 knockdown decreased the percent of cycling cells. Specifically, there was a decrease in S phase cells and an increase in the G2/M population. The S phase decrease could be overcome by BK polyomavirus infection, which is known to push cells into the cell cycle, suggesting that exit from the cell cycle can be overcome. Furthermore, knockdown of p16, a repressor of cell cycle entry also rescued the delay in cell cycle entry as well as viral titres in BKPyV infected primary RPTE cells. Finally, although the co-knockdown of p16 in eS25 knockdown cells rescued S phase and viral titres, a pulse chase analysis revealed that this was the result of an increase in the number of cycling cells from the non-cycling population instead of reversing the eS25 cell cycle block. Therefore, these data suggest that eS25 knockdown results in a robust cell cycle arrest independent of senescence-associated inhibitors, such as p16. The cell cycle arrest resulted in an accumulation of cells in G2/M in the eS25 knockdown cells. These studies suggest that eS25 impacts the cell cycle state. 

HLA Class I antigen processing and presentation (APP) is a highly regulated process that enables CD8+ T cell immunosurveillance. APP begins with the ribosomal synthesis of a source antigen, yet the role of ribosomes, particularly specialised ribosomes, in antigen presentation is poorly understood. Here, Dr Faller will show that the presence of the 'P-stalk' on the ribosome enhances antigen presentation. The addition of the P-stalk to the ribosome is stimulated by cytokines that upregulate APP components, and knockdown of one of the P-stalk proteins (P1) reduces T cell recognition of tumour cells. Mechanistically, he shows that P1-containing ribosomes exhibit enhanced translation of HLA Class I molecules and accessory APP components. Finally, analysis of patient data reveals that the mRNA expression of the P-stalk proteins positively correlates with CD8+ T cell infiltration, a trend not seen for other ribosomal proteins. In all, Dr Faller demonstrates that the presence of the P-stalk defines a specialised ribosome population that enhances antigen presentation, something that may be exploited by cancer cells to escape immunosurveillance.

The dynamic deposition of chemical modifications into RNA is a crucial regulator of temporal and spatial accurate gene expression programs. A major difficulty in studying these modifications, however, is the need of tailored protocols to map each RNA modification individually. In this context, direct RNA nanopore sequencing (DRS) has emerged as a promising technology that can overcome these limitations, as it is in principle capable of mapping all RNA modifications simultaneously, in a quantitative manner, and in full-length native RNA reads. Here, Dr Novoa will present the latest work on how we can use DRS to identify RNA modifications with single nucleotide and single molecule resolution, to then study the biological functions and dynamics of the epitranscriptome, their interplay with other regulatory layers, as well as to decipher how and why epitranscriptomic dysregulation is often associated to human disease.

Rps26-deficient ribosomes, a physiologically relevant ribosome population which arises during high Na+ stress to specifically support the translation of mRNAs involved in the response to high salt stress, are generated via dissociation of Rps26 from fully assembled ribosomes. The chaperone Tsr2 binds ribosomes to directly release Rps26 when intracellular Na+ concentrations rise. Tsr2-mediated Rps26 release is reversible, enabling a rapid response that also conserves ribosomes. However, because the concentration of Tsr2 relative to ribosomes is low, how the released-Rps26 Tsr2 complex is managed, and how Rps26-deficient ribosomes accumulate over time to nearly 50% of all ribosomes, remains unclear. Here, Professor Karbstein shows the existence of a feed-forward loop that enables the accumulation of Rps26-deficient ribosomes in response to prolonged exposure to high salt stress. Her data shows that under high salt stress released Rps26 is degraded via the Pro/N-degron dependent pathway and she has identified the E3-ligases Gid4 and Gid10 as mediators of Rps26 degradation. Moreover, Gid10, which is induced transcriptionally in response to high salt stress also is enriched on Rps26-deficient ribosomes. Finally, substitution of the proline in position 2 of Rps26 to serine increases the stability of free Rps26 and limits the accumulation of Rps26-deficient ribosomes under high salt. Yeast with this mutation, or yeast lacking Gid4 and Gid10 are more sensitivity to high salt stress. Together Professor Karbstein's results demonstrate that degradation of Rps26 released from ribosomes via the Pro/N-degron pathway is important for the generation of Rps26-deficient ribosomes under prolonged salt stress.

Plant acclimation to low temperature occurs through system-wide mechanisms that include proteome changes and altered transcript-to-protein translation. Rather than monolithic executing machines that operate on altered mRNA abundances, ribosomes can contribute to selective translation. Root apices from germinating seedlings of the monocot plant barley were chosen as a model to study changes in protein abundance and synthesis rates during cold acclimation. Knowledge of metabolic and physiological parameters allowed to compare protein synthesis rates in different physiological states, specifically the cold acclimated compared to the optimal temperature state. Professor Kopka shows that during acclimation, specific ribosomal proteins are synthesised and assembled into ribosomal complexes from root proliferative tissue and associate ribo-proteome shifts with changes in other translation-related and non-translation-related macromolecular protein complexes. He works towards understanding how a reconfigured ribosome population may confer selectivity to translation under altered temperature conditions.