The evolution and function of fish TRIM E3 ubiquitin ligases

Project Information

FishTRIM

Grant agreement ID: 101076432

DOI

10.3030/101076432

EC signature date 2 March 2023

Start date 1 September 2023

End date 31 August 2028

Funded under

• European Research Council (ERC)

Total cost

€ 1 499 228,75

EU contribution

€ 1 499 228,75

Coordinated by

UNIVERSIDAD DE SANTIAGO DE COMPOSTELA
Spain

Periodic Reporting for period 1 - FishTRIM (The evolution and function of fish TRIM E3 ubiquitin ligases)

Reporting period: 2023-09-01 to 2026-02-28

Summary of the context and overall objectives of the project

FishTRIM aims to study the evolution and function of the TRIM family of proteins in fish. TRIM proteins are E3 ubiquitin ligases, which in essence means they "decide" which cellular proteins are going to be modified via ubiquitination - the addition of a small protein that leads to changes in protein stability, function or localization. The TRIM family has undergone massive expansions across the fish phylogeny, with single-copy mammalian TRIMs frequently present in large numbers in fish genomes. Additionally, these fish TRIMs seem to be rapidly evolving, with different fish groups showing different TRIM genes. In mammals, TRIM E3 ligases are key in host-pathogen interactions, regulating immune responses and also directly interacting with viral proteins in several cases. Similarly, various fish TRIMs have been associated to antiviral responses, and positive selection has been observed in fish TRIM protein domains involved in protein-protein interactions and pathogen recognition. However, fish TRIMs have been scarcely studied, and the drivers underpinning their expansion(s) in fish is not understood.

The hypothesis underlying FishTRIM is that this family of fish E3 ubiquitin ligases acts as an immune mechanism against specific viral pathogens, and therefore the repeated independent expansions of the TRIM repertoire in fish are the result of an arms race between fish and viruses. To address these questions, FishTRIM will study the function of this family in fish using a variety of approaches, including single-cell genomics to identify the cell types were they are active, genome editing to study their general function within the cells, or proteomics to identify the proteins they are targeting for ubiquitination. These results will be integrated with a phylogenomic approach, where the evolution of the TRIM repertoire across the fish phylogeny will be used to determine potential functional overlap across independent expansions, in addition to identifying selection patterns in the context of TRIM protein domains. Finally, using public databases and AI-powered protein structure prediction, the potential co-evolutionary interactions between fish TRIMs and viral proteins will be evaluated.

FishTRIM will illuminate the function of the expanded TRIM repertoire. The project aims to deliver frontier knowledge on the biological significance of the different fish TRIM expansions, with potential to revolutionise our understanding of fish immune systems and of ubiquitination in host-pathogen interactions. FishTRIM will also explore the role of TRIMs in resistance to viral diseases in fish, with potential transformative outcomes leading to improved fish welfare and food security.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The first two years of the project have been focused on WP1 (evolution of TRIM proteins) and WP2 (function of TRIM proteins.

As part of WP1 we have generated vertebrate-wide phylogenies of the TRIM family. We have focused on the three fish-specific TRIM sub-families, which show massive clade-specific expansions across the whole fish phylogeny. The vertebrate-wide gene trees were too large to present and interpret, and therefore two additional clade-specific analyses were performed in those fish groups with the largest number of genomes: cichlids and acanthuriformes. Gene trees for these subgroups allowed us to better ascertain the rate of birth and death of TRIM genes, which is extremely rapid, with large difference in the TRIM repertoire even between closely related species. This corresponds to deliverable 1.1 “TRIM phylogeny, duplication and loss events”.

To perform this task we had to perform a thorough review of the publicly available fish genomes to ensure only high-quality assemblies were used in our analyses. As a result, we decided to prepare a short manuscript presenting the status of fish genome assemblies. This is currently under review in the journal “Fishes”.

We are now working to finish the remaining analyses in WP1. We have identified signatures of selection across TRIM evolution (Deliverables 1.2 and 1.3) and we are currently putting them in the context of protein domains (Deliverable 1.4). The bioinformatic analysis are near completion, but the interpretation of the results has been challenging due to the sheer amount of information. In any case, the results are quite exciting as they point to strong selective pressures acting across the whole fish phylogeny, with differences depending on the fish-specific TRIM subgroup studied.

In terms of WP2, Tasks 2.1 2.2 and 2.3 are underway and results are expected soon, in line with the planned timeline. In Task 2.1 guide RNA constructs targeting all fish TRIMs in salmon have been designed, and a pooled library of lentiviral vectors has been obtained. We are currently transducing our Cas9-expressing salmon cell line and preparing to build Perturb-seq libraries for single-cell sequencing. In Task 2.2 we have designed and obtained the necessary TRIM-TUBE-FLAG vector for immunoprecipitation of the targets of the fish TRIMs, and we are currently optimizing the immunoprecipitations. Finally, for task 2.3 we are currently interrogating the expression of TRIM genes across development, tissues and immune-activated states in 6 fish species using the datasets generated in the EU project Aqua-FAANG. This information will inform the prioritization of tissues and conditions for single-cell sequencing.

WP3 and WP4 have not started yet, and so far there is nothing to report.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

We have performed CRISPR-Cas9 genome editing in European sea bass and gilthead sea bream cells for the first time. This was not initially planned as part of the project, but arose as an opportunity when immortalised cell cultures of both species were shared with us. For FishTRIM, this represents additional opportunities to study the function of the TRIM repertoire, enabling functional studies in other fish groups beyond salmonids. This should strengthen the robustness of the conclusions of the project. Beyond this ERC Starting Grant, this achievement will facilitate the study of gene function in the two most important species in Mediterranean aquaculture, and will be particularly interesting to study disease resistance.