RSDYN

Ribosome dynamics analysed by novel cross-linking/mass spectrometry

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 Obiettivo del progetto (Objective)

'Ribosomes, large and dynamic particles made of RNA and proteins, are amongst the most complex macromolecular machines known to man. High resolution tools have provided exciting views of the ribosome structure. Revealing dynamics of ribosomal complexes in solution, however, has so far been challenging due to the restricted stability and homogeneity of ribosomes. A set of new tools developed by the Rappsilber lab, partly by me over the last few months, changes dramatically the feasibility of investigating ribosome dynamics. I aim to use these new mass spectrometry-based technologies to determine accurate sites and changes in yield of cross-linking between proteins and between RNA and proteins. Thereby, using protein-protein and RNA-protein cross-linking/mass spectrometry approaches, I aim to answer: 1. Is the final and rate-limiting step in the biogenesis of 50S ribosomal subunits a conformational rearrangement? This will prepare the subsequent analysis of the other steps during the biogenesis of ribosomes in E. coli and in future also in the eukaryote S. cerevisiae. 2. How does the structure and extra-ribosomal protein content of translating ribosomes change in response to different stress conditions? This will complement electron microscopy data and lead into the analysis of how translation links to virulence in pathogenic bacteria.

My project will showcase the potential of cross-linking/mass spectrometry for the study of ribonucleoprotein complexes and, indeed, for protein-nucleotide interactions in general, i.e. encompass DNA-protein interactions. Furthermore, results of this study may facilitate the development of next-generation antibacterial drugs, targeting assembly factors and conformational rearrangements during ribosome biogenesis. For my academic career, acquiring knowledge in cutting edge proteomics approaches will be a stepping stone and further strengthen my role as gatekeeper of proteomics knowledge and a catalyst of biological sciences in Estonia.'

Introduzione (Teaser)

Studying and understanding the assembly of key cellular components is vital for elucidating cell function. European scientists investigated the process of ribosome biogenesis hoping to provide novel anti-bacterial targets.

Descrizione progetto (Article)

Protein synthesis in cells takes place in specialised macromolecular structures called ribosomes made of RNA and protein. Although high-resolution analysis has provided a detailed picture of ribosomal structure, the dynamics of protein-RNA interaction remains largely unknown. This has mainly been due to the instability of complexes in vitro.

Scientists on the EU-funded RSDYN (Ribosome dynamics analysed by novel cross-linking/mass spectrometry) project set out to address this challenge through novel mass spectrometry-based technologies to determine cross-linking between proteins and RNA.

In their experiments, the RSDYN consortium irradiated living yeast cells and identified the part of the peptide that was cross-linked to RNA. Using this approach they precisely mapped the RNA-binding sites of hundreds of yeast proteins. This information was combined with high-resolution analysis of yeast ribosome structures and of individual protein-RNA complexes.

Researches also performed genome-wide analysis of RNA targets of the novel RNA-binding protein enolase. To elucidate protein-protein interactions in ribosome complexes, the consortium used Escherichia coli strains defective in ribosome assembly and studied the alteration in ribosome composition and structure.

The RSDYN method complements existing strategies based on X-ray crystallography and NMR for studying the dynamics of ribonucleoparticles such as ribosomes. This method should be highly useful in many research areas, from basic molecular biology to drug design as well as the study of protein-DNA interactions. Furthermore, the results of this study will facilitate the development of next-generation anti-bacterial drugs that target assembly factors in ribosome biogenesis.