A Riboescaper study: Protein synthesis driven by deficiently assembled ribosomes

Project Information

RiboEscapers

Grant agreement ID: 101024158

DOI

10.3030/101024158

Project closed

EC signature date 19 March 2021

Start date 1 March 2022

End date 29 February 2024

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 160 932,48

EU contribution

€ 160 932,48

Coordinated by

UNIVERSIDAD DE SEVILLA
Spain

Periodic Reporting for period 1 - RiboEscapers (A Riboescaper study: Protein synthesis driven by deficiently assembled ribosomes)

Reporting period: 2022-03-01 to 2024-02-29

Summary of the context and overall objectives of the project

Ribosomes are RNA-protein complexes that translate mRNA into proteins in all living cells. Ribosome biogenesis is the complex process of producing and assembling ribosomes, finely tuned to cellular demands for growth, proliferation, and adaptation.
Additionally, ribosomes are critical parts of eukaryotic cells given that they play an essential function in maintaining protein homeostasis, which requires a precise balance between the processes of protein synthesis, folding, and destruction. In eukaryotes, ribosomes are sequentially generated in key steps where the ribosomal RNA (rRNA) is folded, modified, processed, and assembled with ribosomal proteins (r-proteins) to form the two ribosomal subunits. Recently, the connection of defective ribosome biogenesis with human diseases has become clear. This is the case of the so-called ribosomopathies, human syndromes caused by mutations in genes encoding either r-proteins or ribosome biogenesis factors, which are known to increase the probabilities of cancer development. Although the understanding of the molecular bases of ribosomopathies is rapidly increasing, available treatments (e.g. steroids, bone marrow transplants) are unfortunately scarce, have strong side effects and do not result in a real improvement of the patient's perspective of life.
Considering the multi-steps pathway of ribosome biogenesis, the possibilities to introduce errors with possible harmful effects for cell viability are high. In this context, cells have developed multiple surveillance mechanisms to supervise the structural and functional integrity of the ribosome particle. When a pre-ribosomal subunit suffers an assembly error, in most of the cases, the aberrant particle is retained in the nucleus. However, some aberrant pre-particles have been shown to escape the surveillance mechanisms and to be exported to the cytoplasm.
This project explored the functional consequences of r-protein gene mutations, which generate defective ribosomal particles that are successfully exported to the cytoplasm (RiboEscapers) and actively participate in translation in yeast cell. For this purpose, the research action combined traditional yeast tools together with state-of-the-art methodologies, such ribosome profiling to provide a detailed overview of the r-protein roles in ribosome biogenesis, as well as the physiological consequences caused by specific errors in this process.

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

Our research outlined the physiological effects of ribosome biogenesis deficiencies. We studied null mutants of ribosomal proteins from both ribosomal subunits to: 1) Evaluate their growth and translational capacity, 2) Investigate impacts on protein translation, and 3) Explore potential links to the cytosolic ribosome quality control system
Through rigorous validation, we have established that these proteins are dispensable for cellular viability and that they perform protein synthesis. I have stablished Ribosome Profiling methodology within the host laboratory for the model organisms, Saccharomyces cerevisiae. The application of Ribosome Profiling as well as RNA-Seq methodologies, aimed to elucidate comprehensive insights into the translatome and transcriptome in different yeast strains. All the information generated will be uploaded to the public genomics data webpage Gene Expression Omnibus (GEO), a database repository of high throughput analysis that allows the use of unique identifiers. Among the various unresolved questions, we attempt to elucidate whether aberrant ribosomes exhibit a preference for translating specific pools of transcripts.
In summary of the advancement of the outlined objectives within the action plan and concurrent project commitments, the primary outcomes are summarized as follows: First, physiological characterization of ribosomal mutants from both small and large subunit, namely RiboEscapers, revealed their non-essential role in cell viability.
Second, standardizing growth conditions at 30°C proved conducive for all RiboEscapers.
Third, analysis of ribosomal subunit conditions, polysome assembly, and cellular translation through polysome profiling demonstrated the translation ability of RiboEscapers strains.
Fourth, successful implementation of the Ribosome profiling technique in the host laboratory for Saccharomyces cerevisiae encompassed optimizing cell growth, lysis, nuclease digestion, ribosome isolation, RNA extraction, and footprint generation.
The identity of the ribosomal protein mutants are listed in the fellowship technical report and will be available upon publication.
Following the completion of experimental procedures, we are presently in the stage awaiting the informatics analysis on the deep-sequenced datasets.
The upcoming publications resulting from our ongoing projects will integrate these datasets. Moreover, I am currently engaged in the revision of an article resulting from collaborative actions with Heidelberg University. Additionally, we are preparing a revision within the host laboratory.
During the project, I had the opportunity to present the RiboEscapers project through oral presentations at two national conferences: the IX Spanish Network Meeting on RNA (RIBORED 2022) and the workshop "Elements Empowering RNA Function: From Bench to Bedside".
The project offered extensive training, including proficiency in using Saccharomyces cerevisiae and genetic methods, financial management skills, security training, improved communication through presentations and lectures, mentoring and leadership development by supervising students, strengthened teaching abilities through lectures, and contributing to a successful national grant application
Finally I coordinated the Pint of Science festival in Seville for science outreach. I participated in IBISibiliza's open-door events by the Institute of Biomedicine. As a Marie Curie Ambassador, I gave a presentation on scientific careers at the WOW Science lecture series and another on Rare Diseases for the European Researchers' Night.

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)

The RiboEscapers project addresses a fundamental and unanswered question regarding the functional implications of a quality control failure in ribosome biogenesis. Ribosomes are essential macromolecular machines responsible for protein synthesis in all living cells. Defects in their production and assembly can have severe consequences, contributing to various diseases known as ribosomopathies as well as an increased susceptibility to cancer. The -omic data acquired through the accomplishment of this action will provide an extensive amount of information for subsequent grant applications and projects, with the aim to understand the intricate correlation between errors in ribosome biogenesis and protein translation within the framework of these diseases. By comprehensively analyzing the molecular effects of ribosome biogenesis errors, this project seeks to unravel the mechanistic links between faulty ribosome production and cellular dysfunction. The insights gained from this approach will offer valuable potential pathways for the future development of therapy strategies targeting ribosome biogenesis errors, which could lead to innovative treatments for ribosomopathies, cancer, and potentially other disorders associated with disrupted protein synthesis.

RiboEscapers_model