RiboCode - Unlocking the secrets of specialised ribosomes across eukaryotes

Start date: 1 January 2023
End date: -
Funder: Biotechnology and Biological Sciences Research Council (BBSRC)
Value: £5,600,000
Partners and collaborators: University of Nottingham, University of Sheffield
Primary investigator: Dr Julie Aspden
Co-investigators: Dr Juan Fontana, Dr Antonio Calabrese, Professor Ade Whitehouse, Professor Brendan Davies
External co-investigators: Emma Thomson, Mary O'Connell

Project title

Artistic representation of specialised ribosomes

Description

Co-investigators: Dr Paolo Actis.

Specialised ribosomes are groups of ribosomes that specifically translate a set of mRNAs and represent a new regulatory mechanism in gene expression. This concept is relatively new, and the examples currently described are in single organisms and have limited mechanistic detail.

We aim to understand the detailed molecular mechanisms of specialisation and determine common features across eukaryotes. We will study ribosome specialisation from an evolutionary, functional (translational outcome) and structural perspective, using different eukaryotic model systems (yeast, Drosophila, Arabidopsis, human cells) that cover >1 billion years of evolution. Our team are members of LeedsOmics, the Astbury Centre and the Bragg centre for Materials Research.

We will also develop novel tools, including nanopore-based approaches to characterise specialised ribosomes in single cells.

We will apply these approaches to candidates for ribosome specialisation generated from two strategies:

We have selected 5 examples of specialisation from our preliminary results and the literature;
We will unearth additional novel candidates of ribosome specialisation through evolutionary analyses.

All our results, and those from the literature, will be collated in a public website-based platform, ensuring the legacy of the project.

Overall, this programme of research will allow us to unravel precisely how heterogeneity results in specialisation, and to define a 'ribosome code' applicable to all eukaryotes. In addition to changing our understanding of gene expression, our findings have the potential to impact our understanding of several human diseases.

Ribosomopathies, including Diamond-Blackfan anaemia, are caused by mutations in ribosomal proteins; and it has been suggested that specialised 'oncoribosomes' might be involved in protein synthesis dysregulation in cancers.

Additionally, understanding mechanisms of ribosome specialisation will enable the modulation ribosome translation in future medical, agricultural and biotechnological applications.

This exciting, transformative project will:

Provide an unprecedented understanding of how specialised ribosomes regulate translation at the molecular level through changes in their composition.
Reveal the critical sites of ribosome specialisation across eukaryotes.
Generate a ‘ribosome code’ to explain the observed common mechanistic features of how specialised ribosomes regulate translation across eukaryotes.
Employ the ‘ribosome code’ to predict additional routes of specialisation to provide the potential for future engineering of gene expression.

Our objectives

Together we will address this problem via four interlinked objectives, delivered through their corresponding work-packages (WPs):

Obj-1) Identify potential sites of specialisation and determine common patterns of specialisation across eukaryotes.

Obj-2) Determine which mRNAs are translated by various specialised ribosomes.

Obj-3) Assess how variations in ribosome composition structurally regulate translation.

Obj-4) Develop novel biophysical tools to probe ribosome specialisation.

Objective 1

Identify potential sites of specialisation and determine common patterns of specialisation across eukaryotes.

In our comparative genomics workpackage we are focussed on uncovering common routes to specialisation across all of eukaryotic life - cracking the “ribosome code”.
We will generate hypotheses of sequence driven specialisation using conserved evolutionary features in RPs and associated proteins. This will be achieved using novel phylogenomics approaches designed for this project and large scale genome data analytics. We will extract key evolutionary features from the sequences of RPs and associated proteins identifying levels of conservation of these features across all evolutionary depths from eukaryote-conserved to Kingdom-conserved (Plants, fungi, Animals) and within Kingdom-conserved. Integrating data from across the program of research, and combining traditional comparative genomics and phylogenomics with machine learning techniques, we will generate hypotheses of ribosomal specialisation which the sLoLa team will test from structural and functional perspective.

Objective 2

Determine which mRNAs are translated by various specialised ribosomes.

The translational work package will investigate the functional and mechanistic conservation of specialised ribosome components, in each of our 5 model systems. Central to both assessing a ribosome population for specialisation and dissecting their molecular mechanism of regulation is understanding specific translational output. To do this we will engineer cell lines and organisms with affinity tagged specialized ribosome components to enable selective RiboSeq. Riboseq will determine which mRNAs are regulated by specialised ribosomes and provide information on how this regulation may be achieved. This will allow the same “type” of specialized ribosomes to be compared across multiple organisms and provide experimental evidence of functional conservation. These analyses will also allow the identification of conserved features of mRNAs which are targeted by specialised ribosomes in each model, which will inform subsequent functional testing. The role each specialised component plays in translation and its conservation will be assessed through mutational analyses, translational reporters and cross species complementation assays.

Objective 3

Assess how variations in ribosome composition structurally regulate translation

Central to dissecting the common mechanisms of translational regulation by specialised ribosomes, and something currently missing for most specialised ribosomes, is an understanding of the structural impact of specialisation on translation regulation. This will be addressed in the structure of specialised ribosomes WP, which, combined with the evolutionary analyses and the translational output, will allow us to unravel the molecular mechanisms of ribosome specialisation.Our established structural approach to characterise specialised ribosomes involves: 1) Purifying candidate ribosomes and determining their protein composition, including the identity and stoichiometry of protein subunits that confer specialisation, by quantitative MS (e.g. TMT-MS). And 2) Characterising their structure by cryo-EM, to provide insight into the structural differences that drive specialisation. This workflow has allowed us to unravel composition and structural differences in ribosomal paralogs between Drosophila ribosomes isolated from testes and ovaries, and we will use it during the sLoLa to gain structural information on the specialised ribosomes we will purify. To obtain further information on regions of the ribosome that have been historically challenging, we will adapt structural MS approaches for the study of ribosomes, including native MS, which allows unravelling the stoichiometry and composition of specialised ribosomes, and cross-linking MS, which allows probing for structural/dynamical changes in the ribosome.

Objective 4

Develop novel biophysical tools to probe ribosome specialisation.

Transformative technologies:
The aim of this WP is to develop a range of new biophysical tools to probe ribosome specialisation. For example, we will develop novel tools to enhance the functional analysis of ribosomal complexes and to complement and enrich the structural characterisation delivered by WP3.

One of the challenges in dissecting specialised ribosomes is small sample size, given their

tissue/developmental time-point specific formation. We will develop a solid-state nanopore platform to tackle this limitation by enabling ribosome analysis with single entity resolution (Raveendran et al, ACS Sensors, 2020).

To test the ability of specialised ribosomes from one species to regulate translation in another, we will develop a nanoinjection platform based on nanopipettes to introduce ribosomes into naïve cells to investigate any translation reprogramming using state-of-the-art single cell proteomics approaches.

Finally, we will generate a database platform which integrate the data types for specialised ribosomes in different species across all WPs. The database will be made freely available to the scientific community to maximise the impact of our results.