Study On The Screening Of Interacting Proteins With Glutamine TRNA Synthetase Splice Variant QRS-C13

Aminoacyl-tRNA synthetases(AARSs)are the enzymes that catalyze the aminoacylation reaction by covalently linking an amino acid to its cognate tRNA in the protein synthesis.Beyond this classical function,its also have many nonclassic functions.glutamine tRNA synthetase(Gln RS,QRS)is one of the Aminoacyl-tRNA synthetases.glutamine tRNA synthetase has a splice variant QRS-C13,it lacks the core catalytic domain of glutamine tRNA synthetase,thus losing its catalytic function.Which proteins interact with QRS-C13?What 's the role does QRS-C13 play in its function? By comparing the differences between QRS-C13 binding proteins and QRS-C13 binding proteins,we screened proteins that specifically interact with QRS-C13.This is important for understanding the function and mechanism of QRS-C13 and discovering the non-classical functions of QRS.In this study,recombinant plasmids p IRESpuro2-QRS-C13 and p IRESpuro2-QRS were transfected into Hela cells.We found that there was a great difference between the QRS-C13 binding protein and the QRS binding protein by co-immunoprecipitation,and the specific proteins were selected.Mass spectrometry analysis revealed possible interaction between QRS-C13 and Eukaryotic translation initiation factor 3 subunit L(e IF3L)and leucyl-tRNA synthetase(LRS).The interaction between QRS-C13 and e IF3 L was verified by Western-blot.Recombinant plasmids p ET20b-QRS-C13,p GEx-4T-2-e IF3 L,and p GEx-4T-2-LRS were successfully expressed in E.coli to obtain QRS-C13,e IF3 L and LRS proteins.GST-pull down experiments revealed that the interaction between QRS-C13 and e IF3 L was an indirect interaction In vitro,and there was a direct interaction between QRS-C13 and leucyl-tRNA synthetase(LRS),and there was a direct interaction between e IF3 L and LRS.Therefore,we speculate that the interaction between QRS-C13 and e IF3 L the participation of LRS,and that QRS-C13,e IF3 L,and LRS may form some form of ternary complex or part of a multiplex complex.Through cell localization experiments,it was found that QRS-C13 mainly concentrates in the nucleus,and e IF3 L mainly concentrates in the cytoplasm.We speculate that QRS-C13 may be involved in related life activities in the nucleus.The results of this study laid the foundation for the study of the function of QRS-C13,and also provided new ideas for the research of non-classical functions of QRS.