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Overview

Scientific discussion meeting organised by Dr Julie Aspden, Dr Maria Barna, Dr William Faller, and Professor Anders Lund.

Ribosomes were thought to be homogeneous, passively translating mRNA into proteins. However, recent work has discovered that the composition of ribosomes is highly heterogeneous and that different ribosome populations can regulate the translation of specific mRNAs. This meeting will discuss latest advances, covering all types of ribosome heterogeneity in a variety of organisms and systems, including impact on human disease.

The schedule of talks, speaker biographies and abstracts will be available soon. Meeting papers will be published in a future issue of of Philosophical Transactions of the Royal Society B

We particularly encourage the participation of early career researchers (PhD students, postdocs and research technicians) to submit abstracts for either the short talk or the poster session on their latest work on ribosome heterogeneity and specialisation. Work can cover any type of ribosome heterogeneity in a variety of organisms and systems, including impact on human disease.

Short talk from abstract

The organisers invite researchers in the field to submit a short abstract (200-250 words) to be selected for a presentation slot within the meeting. If successful, you will be required to prepare a 10 minute presentation which will be followed by 5 minutes for questions and discussion. Please submit your presentation title and abstract to the Scientific Programmes team no later than Friday 22 September. Your submission should include the text 'Short talk abstract submission' in the email subject line.

Poster session

There will be a poster session on Monday 6 November. If you would like to present a poster, please submit your proposed title, abstract (no more than 200 words and in third person), author list the name of the proposed presenter and institution to the Scientific Programmes team no later than Friday 6 October 2023. Please include the text 'Poster abstract submission' in the email subject line. The organisers also encourage online participants to submit a poster abstract. If successful, they will be displayed on the online event platform for the duration of the meeting.

Attending this event

This event is intended for researchers in relevant fields.

  • Free to attend
  • Both in person and online attendance available
  • Advance registration is essential. Registration is now open. 

Enquiries: contact the Scientific Programmes team.

Organisers

Schedule


Chair

09:05-09:30
Biochemical and genetic characterisation of ribosome biogenesis and functional diversity

Abstract

Baker’s yeast ribosomes are composed of four structural RNAs and 79 proteins that are selected from a panoply of ~150 ribosomal RNA (rRNA) genes and 137 ribosomal protein genes (RPGs). Most RPGs are duplicated (dRPGs) and the selective pressure maintaining these gene-duplications and their functional significance remain largely unexplored. It was initially believed that RPGs have remained duplicated over the aeons to satisfy the high demand for ribosome production by providing a sufficient dose of duplicated protein paralogs with identical functions. However, our studies of ribosome production revealed that most ribosomal protein paralogs are independently regulated and that they respond to different growth conditions. We proved this by separately deleting the duplicated paralogs and observing distinct phenotypic effects and functions that could not be complemented by their evolutionary partner genes. As a clue to the evolved functional divergence, we found that the ratio of expressed dRPGs depends on growth conditions, leading to ribosomes with altered make-ups and consequently to changes in the program of mRNAs that are selected for translation. Together these observations argue against an equal and redundant role for dRPGs and propose a new model where each paralog is uniquely regulated to serve a particular function during ribosome biogenesis and protein synthesis.

Speakers

09:30-09:55
Building a case for 'functional identity fraud' in eRpL22 paralogue-specific ribosomes in Drosophila germline development

Abstract

Co-expression of eukaryotic-specific ribosomal protein paralogues, eRpL22 and eRpL22-like, within the male germline generates heterogeneous ribosomes are shown by paralogue-specific polysome immunoprecipitation and mRNA characterisation by RNAseq to translate distinct mRNAs. Dr Ware developed a model Drosophila S2 cell system that recapitulates the enrichment pattern for model mRNAs detected on eRpL22-like ribosomes in the testis to explore the mechanism of targeted translation. The mechanism for targeting specific mRNAs to- or excluding mRNAs from- paralogue-specific ribosomes is unknown. For targeted mRNAs, the structural characteristics governing association remain unclear. No clear consensus structural features are detected among enriched mRNAs shown by GO analysis to function in the same biological process. Additional computational analyses may uncover conserved features for further study. On the paralogue side, eRpL22 and eRpL22-like are structurally distinct, most notably at the N terminus that projects from the subunit surface. 60S subunit models position eRpL22/eRpL22-like on the solvent side at the subunit base, removed from the subunit interface.  eRpL22/eRpL22-like N- and C- terminal extensions are theoretically available for interactions that could create an interface between the base and the preinitiation complex. Dr Ware hypothesises that the N terminal extension is required for specific mRNA enrichment on eRpL22-like ribosomes. She is using the S2 system transfected with constructs lacking the eRpL22-like N-terminal domain to quantify and compare model mRNA association with engineered eRpL22-like ribosomes to mRNAs associated with wild type eRpL22-like ribosomes. These experiments will provide insights into the potential role of the N terminus of eRpL22 paralogues in specifying specific translation.

Speakers

09:55-10:20
Tbc

Abstract

Abstract available soon.

Speakers

10:20-10:30
Discussion
10:30-11:00
Break
11:00-11:25
Neuronal ribosome localisation and heterogeneity

Abstract

Owing to their morphological complexity and dense network connections, neurons modify their proteomes locally, using mRNAs and ribosomes present in dendrites and axons. Professor Schuman will discuss the group's efforts to understand the neuronal ribosome population present in dendrites and axons, including work on the abundance and dynamics of ribosomes.

Speakers

11:25-11:50
Exploring the function and structure of heterogeneous ribosomes in the gonads of Drosophila melanogaster

Abstract

Dr Aspden has previously profiled the composition of ribosomes from different Drosophila tissues. She discovered the enrichment of specific RP paralogs within the ribosomes of Drosophila gonads, with 4 paralogs enriched in ovary and 6 in testis. One of these testis-specific paralogs, RpL22-like, is incorporated into ~50% of ribosomes in the testis and shares 45% aa sequence identity with the canonical paralog, RpL22. 

To assess the impact of RpL22/RpL22-like paralog switching RNAi was performed. RpL22 knockdown in the ovary affected germ cell maintenance, while knockdown in the testis had no observable effect. This ovary phenotype was not fully rescued by ectopic expression of RpL22-like. RpL22-like RNAi did not impact spermatogenesis and had no effect on the ovary. Immunostaining has revealed that RpL22 and RpL22-like localise to different cellular subtypes and different sub-cellular compartments within the testis. Together these experiments suggest that RpL22 and RpL22-like possess different functional roles during gametogenesis.

To dissect the contribution of RpL22-like to mRNA translation RNAi followed by quantitative mass spectrometry was performed. The levels of 18 proteins change upon knockdown, suggesting that the incorporation of RpL22-like may alter the ribosome’s translational preference. Levels of DGP-1 protein, an ortholog of mammalian ribosome rescue factor, GTPBP1, were reduced ~50% upon RpL22-like RNAi, while mRNA levels were unaffected. This suggests that RpL22-like may translationally regulate DGP-1 mRNA. Work is underway to understand the mechanism by which RpL22 and RpL22-like containing ribosomes regulate the translation of specific mRNAs, and how specialised ribosomes contribute to gametogenesis.

Speakers

11:50-12:05
Short talk from abstract

Abstract

Abstract available soon.
12:05-12:20
Short talk from abstract

Abstract

Abstract available soon.
12:20-12:30
Discussion
13:30-14:30
Keynote: From origin of life to next generation RNA-based therapeutics

Abstract

The site for peptide bond formation in the ribosomes, the PTC, is located within a highly conserved internal pocket made exclusively of rRNA. The high conservation implies its existence irrespective of environmental conditions and indicates that it may represent a prebiotic RNA machine, which could be the kernel around which life originated. Lab constructs imitating this pocket possess capabilities for peptide bond formations, thus indicating that a molecular prebiotic bonding entity still exists and functions within ribosomes of all living cells. In contrast, other ribosomal components undergo genetic variability that led to the creation of specific structural features. Among them, those related to ribosomal genetic diseases, or specific to antibiotics resistant pathogens, are being used as bases for the design of RNA based next generation therapeutics.   

Speakers

14:30-14:45
Short talk from abstract

Abstract

Abstract available soon.
14:45-15:00
Short talk from abstract

Abstract

Abstract available soon.
15:00-15:30
Break
15:30-15:55
Endogenously encoded ribosomal RNA Sequence variation can regulate gene expression and phenotype

Abstract

Abstract available soon.

Speakers

15:55-16:20
Transcript- and species-specific translation by ribosome expansion segments

Abstract

Ribosomes have recently emerged to directly regulate gene expression. Roles for ribosomal RNA (rRNA) in gene regulation remain largely unexplored. Expansion Segments (ESs) are rRNA regions exposed on the outer ribosomal shell that consist of many highly variable, tentacle-like rRNA structures that extend from the conserved rRNA core in eukaryotes; with largely unknown roles in translation. Dr Leppek found a role for ESs in mRNA-specific binding and translation. She identified a single 18S rRNA ES, ES9S, as the interaction site for an Internal Ribosome Entry Site (IRES)-like RNA structure of the Homeobox a9 (Hoxa9) 5’ UTR that directs spatiotemporal, tissue-specific translation control of HOXA9 expression in the mouse embryo. As ESs are highly variable across evolution, Dr Leppek engineered chimeric, 'humanised' yeast ribosomes for ES9S in the yeast 18S rRNA, that endow reconstituted, species-specific rRNA ES binding of 5’ UTR motifs. She next developed VELCRO-IP (variable expansion segment-ligand chimeric ribosome-IP) RNA-seq to site-specifically map the interactome of mRNA regions in the embryo transcriptome that preferentially associate with hES9S. This work identified multiple hES9S-selective mRNAs that undergo cap-independent translation control. Dr Leppek also confirmed IRES-like activity of Hox IRES-like elements with circular RNA reporters. Further, the Hox stem-loop RNA top-ranked in her unbiased screen of mRNA features for optimised RNA therapeutics. This work unravels species-specific, selective mRNA-rRNA interactions on the ribosome for translation. Dr Leppek's own lab focuses on how rRNA-directed specialised translation is employed in the innate immune response.

Speakers

16:20-16:30
Discussion
16:30-17:00
Poster flash talks
17:00-18:00
Poster session
09:00-09:25
rRna modifications in health and disease

Abstract

Abstract available soon.

Speakers

09:25-09:50
Translatome analysis of cancer associated ribosomal protein mutations

Abstract

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.

Speakers

09:50-10:15
Tuning the ribosome in health and disease?

Abstract

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.

Speakers

10:15-10:30
Short talk from abstract

Abstract

Abstract available soon.
10:30-11:00
Break
11:00-11:25
The nonessential ribosomal protein eS25 is required for the cell cycle

Abstract

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. 

Speakers

11:25-11:50
The ribosomal P-stalk regulates HLA Class I antigen presentation

Abstract

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.

Speakers

11:50-12:15
Decoding the ribosomal epitranscriptome and its dynamics at single molecule resolution

Abstract

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.

Speakers

12:15-12:30
Discussion
13:30-13:55
Making Rps26-deficient ribosomes

Abstract

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.

Speakers

13:55-14:20
Towards analysis of remodelled ribosomes in proliferating plant tissue during cold stress

Abstract

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.

Speakers

14:20-14:45
Regulation of ribosome biogenesis and translation by RNA modifications

Abstract

Abstract available soon.

Speakers

14:45-15:00
Discussion
15:00-15:30
Break
15:30-15:45
Short talk from abstract

Abstract

Abstract available soon.
15:45-16:00
Short talk from abstract

Abstract

Abstract available soon.
16:00-16:15
Discussion
16:15-17:00
Panel discussion