RiboCode - Unlocking the secrets of specialised ribosomes 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.

 

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.

 

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.

 

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.