Description du projet
Faire la lumière sur le processus cellulaire de la biogenèse des ribosomes
La biogenèse des ribosomes est le processus de création des ribosomes, qui sont responsables de la qualité et de la quantité des protéines contenues dans les cellules. Il s’agit d’un processus central dans chaque cellule en croissance. L’assemblage défectueux des ribosomes est considéré comme pathogène car il conduit à de nombreuses maladies. Le projet Human‑Ribogenesis, financé par l’UE, fera la lumière sur le mécanisme de biogenèse des ribosomes dans les cellules humaines, ce qui nous aidera à mieux comprendre son lien avec les voies de régulation et la maladie. Plus précisément, il fournira un inventaire structurel presque complet de la biogenèse des ribosomes humains en utilisant la microscopie cryoélectronique de pointe sur des particules préribosomiques natives purifiées. Des informations supplémentaires seront obtenues par l’analyse chimique de la spectrométrie de masse à liaisons croisées, le séquençage des nanopores et la cryotomographie.
Objectif
The emergence of numerous diseases caused by defective assembly of ribosomes calls for a deeper understanding of human ribosome biogenesis. Yet, in human cells the assembly of ribosomal subunits is a vital process of daunting complexity, which requires several hundreds of assembly factors (AFs). Together with small nucleolar RNAs (snoRNAs) these factors facilitate a cascade of modification, processing and folding events of the ribosomal RNA (rRNA) that are tightly coordinated with the incorporation of a large set of ribosomal proteins between the nucleolus and the cytoplasm. This process has been extensively studied in fungal model organisms such as S. cerevisiae, however, assembly and rRNA processing pathways differ significantly between yeast and mammals. Many unique AFs have been identified in humans and several diseases, so-called ribosomopathies, have been assigned to ribosome biogenesis defects. Yet, so far only a single publication (Ameismeier et al., 2018) provides direct molecular information on the structural basis and architectural transitions of human ribosome maturation. Therefore, I propose to provide a near complete structural inventory of human ribosome biogenesis by using state-of-the-art cryo-electron microscopy (cryo-EM) on purified native pre-ribosomal particles. Corresponding analysis by chemical cross-link mass spectrometry, Nanopore sequencing and cryo-tomography as well as functional assays probing for nuclear export and RNA processing defects will gain complementary functional information. Finally, shot-gun cryo-EM of total pre-40S, pre-60S and 90S intermediates will be established in order to quantitatively characterize the equilibrium flow of ribosome assembly in normal and challenged human cells. Together, these insights will provide the basis for a mechanistic understanding of human ribosome biogenesis and will thereby lay the foundation for better relating this process to regulatory pathways and disease.
Champ scientifique
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteins
- natural sciencesbiological sciencesgeneticsRNA
- natural sciencesbiological scienceszoologymammalogy
- natural scienceschemical sciencesanalytical chemistrymass spectrometry
- natural sciencesbiological sciencesmolecular biologystructural biology
Mots‑clés
Programme(s)
Thème(s)
Régime de financement
ERC-ADG - Advanced GrantInstitution d’accueil
80539 MUNCHEN
Allemagne