Group leader: Marc Mirande
Multi-aminoacyl-tRNA synthetase complexes
In higher eukaryotic cells, several aminoacyl-tRNA synthetases are always found in multi-enzyme complexes of apparent molecular masses comprised between 0.7 and 1.5 MDa. Two types of stable macromolecular assemblies have been described from a variety of mammalian sources. The VEGA complex (Valyl-tRNA synthetase/Elongation factor 1A/Guanine exchange factors Assembly) associates a single aminoacyl-tRNA synthetase to the four subunits EF1A, EF1B(alpha), B(beta) and B(gamma) of the translation elongation factor 1. The MARS complex (Multi-Aminoacyl-tRNA Synthetase) is composed of the glutamyl-, prolyl-, isoleucyl, leucyl-, methionyl-, glutaminyl-, lysyl-, arginyl-, and aspartyl-tRNA synthetases, and of the three auxiliary proteins p43, p38 and p18. Its apparent molecular mass is close to 1.5 MDa. This type of structural organization is restricted to eukaryotic species ranging from arthropods to mammals, representing the metazoan phylum of coelomate species. The emergence of multi-enzyme assemblies is accompanied by the evolutionary acquisition of discrete protein-protein interaction motifs, of a scaffold protein (p38), and of a multi-catalytic polypeptide (Glutamyl-Prolyl-tRNA synthetase; or EPRS). Additional tRNA-binding domains are also grafted onto some of these enzymes or on the multi-AARS complex via association of non synthetase components. All these features have strong implications on the cellular organization of translation in higher eukaryotic cells.
The major objective orf our research program is to provide a comprehensive view of the cellular organization of the protein synthesis machinery in the eukaryotic cell. Several aspects are considered. A detailed structural and functional study of the macromolecular assemblies that are specific landmarks of the translational machinery in eukaryotes is conducted. A pluridisciplinary approach is undertaken, using the tools of biochemistry, enzymology, molecular, cellular and structural biology. The process of complex assembly is analyzed in vitro but also in cellulo. The function of the polypeptides extensions and of the auxiliary components associated with aaRSs is examined by using purified recombinant proteins, but also by in cellulo methodologies involving overexpression or silencing approaches.
Lysyl-tRNA synthetase and HIV-1 replication
The primer for reverse transcription of the HIV-1 genome is tRNA(Lys,3). During assembly of HIV-1 particles, tRNA(Lys,3) is taken up along with LysRS from the host cell, the tRNA binding protein that specifically aminoacylates the different tRNA(Lys) isoacceptors. In human, the cytoplasmic and mitochondrial species of LysRS are encoded by a single gene by means of alternative splicing. Polyclonal antibodies directed to the full-length cytoplasmic enzyme equally recognized the two enzyme species. We raised antibodies against synthetic peptides that allowed to discriminate between the two enzymes and found that mitochondrial LysRS is the only cellular source of viral LysRS. Mitochondrial localization of LysRS in HeLa cells is altered after addition of the auxiliary viral protein Vpr in the culture medium. These results open new routes toward the understanding of the molecular mechanisms involved in the specific packaging of tRNA(Lys,3) into viral particles.