The team “RNA Molecular Machines and human pathlogies ” specialises in structural biology of proteins and RNA, and of their complexes with small molecules.
Our group is involved in three main research themes:
(I) Structural biology of macromolecular complexes,
(II) structural studies of protein-inhibitor complexes
(III) high-resolution cristallochemistry of small molecular compounds.
The area of expertise of the team is in biochemistry and structural biology techniques: protein and RNA production and purification, structural analysis (crystallography, SAXS, cryo-electron microscopy…) and functional assays (biophysical and biochemical interaction assays, fluorescence, enzymology,…)
1 – Integrative structural and functional biology of RNA Molecular Machineries
Our main project is aimed at deciphering the molecular mechanisms of RNA molecular machines.
Ribonucleoproteins (RNPs) are complexes composed of proteins and RNA molecules that play crucial roles in the cell. These RNPs are involved in a wide range of cellular and biochemical functions, from maturation of different classes of RNAs to protein synthesis, and represent some of the most complicated molecular machines. Our interest lies in understanding how these molecular machines are assembled and regulated in the cell.
During the last we have contributed to the understanding of RNP biogenesis using structural biology to determine the structure of proteins involved in these processes. We have targeted several different aspects of RNP maturation with a focus on small protein complexes which are autonomous building blocks of the RNP. Where possible, we have also tackled protein/RNA complexes, when the RNA binding site is known.
The determined structures have lead to functional hypotheses which we have tested in vivo in the yeast model system. Our results have helped to understand the molecular basis of some genetic disorders (ribosomopathies) and help the development of therapeutic drugs.
2 – Ribosome biogenesis, a promising target in cancer therapies.
During tumorigenesis, cancerous cells need to proliferate. One of the first changes in the cell program is the deregulation of ribosome biogenesis, that leads to greater amount of ribosome and increased translation. The last years have seen the discovery of a tight link between ribosome biogenesis and cell cycle control, with mechanisms involving the direct interaction of ribosome assembly factors with p53 regulon. Several studies have shown that inhibition at different levels of ribosome biogenesis leads to the stabilization and accumulation of p53, and that this regulation was disrupted in cancerous cells.
The project lies on the hypothesis that targeting the ribosome biogenesis/p53 (RB/p53) regulation pathway is an attractive and complementary alternative to targeting the p53/hDM2 interaction for anticancer therapies. We aim to contribute to the understanding of the regulation of p53 by ribosomal proteins and ribosome assembly factors and build on our previous results to propose molecules which impact the RB/p53 pathway. These drugs will have a dual therapeutic interest in the treatment of cancer by activating the tumour suppressor p53 and affecting ribosome synthesis in cancers where p53 is inactivated.
How hDM2 activity by association with ribosomal proteins is regulated at molecular level remains elusive. will use all the techniques of integrative structural biology toolbox to study the human RB/p53 regulon to complete the molecular details of the interaction between hDM2, p53, ribosomal proteins and other ongenic factors. Besides biological insight, this could provide a structural canvas for the design of novel molecules targeting this pathway.
3 – Protein-inhibitor complexes
Our activity in structural biology frequently brings to us collaborations from biologists and chemist who want to obtain structural information on their biological target or its interaction with novel therapeutic compounds. We are open to collaborations where we are implicated at various levels: protein cloning, production and purification, binding studies and/or structure solution of the protein of interest in complex with small molecular compounds.
Our activity in structural biology frequently brings to us collaborations from biologists and chemist who want to obtain structural information on their biological target or its interaction with novel therapeutic compounds. We are open to collaborations where we are implicated at various levels: protein cloning, production and purification, binding studies and/or structure solution of the protein of interest in complex with small molecular compounds.
4- Highlights
4 -1- RNA mimicry in ribosome biogenesis
The Fap7 ribosome assembly factor is involved in regulates ribosomal RNA processing and is essential for embryogenesis and tumour growth, most probably through the ribosomal protein-hDM2-p53 pathway. Fap7 is particularly intriguing because it interacts with the small subunit ribosomal protein Rps14 and it exhibits adenylate kinase activity – a molecular function more commonly associated with regulating ATP/ADP levels than assembling protein–RNA complexes. Combining crystallography, SAXS and biochemical analysis of the Rps14–Fap7 complex, we showed that Fap7 uses RNA mimicry: protein side chains mimic RNA contacts, rendering the interaction of Rps14 with ribosomal RNA or with Fap7 competitive and mutually exclusive. We were able to propose a model in which Fap7 uses RNA mimicry to temporarily remove Rps14 from the ribosome and enables a conformational change of the ribosomal RNA that is needed for the final maturation step of the small ribosomal subunit (Loc’H et al., PloS Biology, 2013).
4 -2- Chaperoning RNA assembly on pre-ribosomes
The 5S RNA is a central player in the activation of the p53 pathway upon nucleolar stress and cancerogenesis. The 5S rRNA is transcribed by RNA polymerase III and is assembled into the 5S ribonucleoprotein particle (RNP), containing ribosomal proteins Rpl5 and Rpl11, prior to its incorporation into preribosomes. The assembly of the 5S RNP into preribosomes is performed by a specialized complex composed of Rpf2 and Rrs1. We have solved the structure of the Rpf2-Rrs1 complex alone, in solution with 5S RNA and placed the structure within cryo-EM maps of pre-ribosomes (T1-20). We showed that the Rpf2-Rrs1 complex establishes a network of interactions that guide the incorporation of the 5S RNP in preribosomes in the initial conformation prior to its rotation to form the central protuberance found in the mature large ribosomal subunit. In order to decipher a precise ribosome assembly step, we used crystallography, SAXS, cryo-EM as well as biochemical protein-RNA binding assays in vitro and in vivo (CRAC) (Madru et al., Genes Dev. 2015).
4 -3- Deciphering the function of molecular motors
RNA helicases are fascinating molecular motors capable of separating double stranded RNA duplexes. Our model protein is Prp43, a remarquable helicase because it is involved in different biological processes (ribosome biogenesis, splicing,…) and requires protein co-factors for its in vitro and in vivo function. We try to understand the molecular details of how these nanomotors work and how they are regulated by protein co-factors. We have already discovered a new intriguing coupling between the nucleic unwinding function and nucleotide base binding (Robert-Paganin et al., NAR, 2017). We are currently investigating the function by single molecule assays in collaboration with V. Croquette (ENS Paris).