Étude de la dynamique des interactions des constituants du complexe de biosynthèse et d’insertion de la sélénocystéine dans la traduction des sélénoprotéines in vivo
Selenoproteins are proteins that incorporate selenocysteines, which is called the 21st amino acid, during their translation. Specifically, selenocysteine (Sec) is a metabolic derivate of serine, which is structurally equivalent to Cys except for replacement of sulfur with an atom of selenium in the δ-position. It differs from other amino acids since a unique synthetase converts the serine into Sec while the residue is already attached to the tRNA. The codon for Sec on the mRNA is a UGA codon that is usually a STOP signal, introducing the concept of "recoding". Through a specific metabolic machinery for the tRNASec and the presence of a SecIS (Selenocysteine Insertion Sequence) on the mRNA, this codon allows the incorporation of the Sec into the protein. However, the mechanism of biosynthesis of this amino acid and its incorporation into proteins is not well understood. It is known that the synthesis starts with the acetylation of tRNA[Ser]Sec by seryl-tRNA synthetase (SerRS) to give the seryl-tRNA[Ser]Sec. The latter is subsequently phosphorylated by the O-phosphoseryl-tRNA[Ser]Sec kinase (PSTK) which will generate the O-phosphoseryl-tRNA[Ser]Sec. Subsequently, a large complex of several proteins and cofactors acting as machinery for incorporation of Sec during translation, associates with tRNA[Ser]Sec and the mRNA and finally, the components of the ribosome. Among these proteins, SepSecS catalyzes the final step in the biosynthesis of Sec converting O-phosphoseryl-tRNA[Ser]Sec into selenocysteinyl-tRNA[Ser]Sec using monoselenophosphate as a source of selenium. Recent studies showed that the association with SECp43 would be required for SepSecS to play its role to segregate into the nucleus to be associated with the machinery that recognizes the UGA codon. Among the proteins of the biosynthesis machinery of selenoproteins that we analyzed, there are eEFSec, RPL30, SPS2, SPS1, SBP2 and NSEP1. Results of analysis of the dynamics of the interaction between the components of the Sec biosynthetic machinery confirm some data from the literature, but conflict with the model so far established. A better understanding of the dynamics of interactions between its constituents and the reaction of this process would allow us to make assumptions about the role of the biosynthetic machinery of selenoproteins and the importance of its complexity. We analyzed the dynamics of interaction in vivo in HEK293T cells using a Protein-Fragment Complementation Assay (PCA) based on a humanized Renilla luciferase (hRLuc) by coupling, by molecular cloning, genes encoding each protein of interest with gene encoding fragments of the luciferase protein (hRluc). We have thus achieved an expression of fusion of N-terminal and C-terminal fragments for each luciferase protein of interest. Then, we analyzed the dynamics of the interaction with the components of the Sec biosynthetic machinery. Further work will be essential to build on the results presented in this research.
eEFSec NSEP1 PCA SBP2 SECp43 SepSecS SLA/LP Protein fragment complementation assay RPL30 SPS1 SPS2