Effects Of Glutamine TRNA Synthetase With Ser⁷⁰,Lys³⁹⁴ And Tyr⁴⁹¹ Mutants On The Stabilityof The Aminoacyl-tRNA Synthetase Complex

Aminoacyl-tRNA synthetase(AARS) is the key enzyme in the process of the protein synthesis.The canonical function of AARS is to catalyze the attachment of amino acids to their cognate tRNAs and compose the aminoacyl-tRNA.The recent stduies show that the AARS couples with related proteins,and form a huge polycomplex to realize its complex biology function,such as the synthesis of rRNA, cell apoptosis, translation regulation, transcription regulation, signal transduction and other noncanonical function.Therefore,AARS play an important role in the cell survival,and it is also closely related with human cancer and other diseases. Glutamyl tRNA synthetase(QRS) is a kind of aminoacyl-tRNA synthetase, and it compose the subcomplex with RRS and P43 in the complex.At the same time,it has a tighter function with RRS and P43 than other AARS.Besides canonical function that it translates the protein,it has the anti-apoptosis function rely on the glutamine in the cancer cells.For these reasons,it is significant to study the conformation,function and stability of the QRS.The purpose of this study is to investigate the correlations between the amino acids locus of glutamyl-tRNA synthetase and the stability of forming AARS complex which i s of great significance for further research on the functions of glutamyl-tRNA synthetase.This study designed three phosphorylation mutation sites,by creating eukaryotic expression vector pIRESpuro2-Tap-QRS and abrupt changed the QRS’s 70 th amino acid residue which should be Ser to Glu,the 394 th Lys mutanted to Arg and the 491 th Tyr mutanted to Phe.Then the QRS-WT and mutants was transiently transfected into HELA and HEK293 cells,compared the effects that the mutants have on the AARS complex.First of all,immunoprecipitated(IP) was used to separate QRS-WTs and mutants from the cell lysis supernatant.Then take the mentioned protein uesd Western blotting(WB) with antibodies raised against Tap and RRS,As a result,there was no obvious difference between the QRS-Tap and RRS from the QRS-WT and QRS-Ser70 mutant,however there was between the QRS-Tap and RRS from the QRS-Lys394 and Qrs-Try491. Next we used MTT to detect the actibity of cell apoptosis in QRS-WT and mutant,it shows that the QRS-Lys394 and QRS-Tyr491 have higer activity than the widetype.The result indicates that the phosphorylation mutation site of catalytic domain in QRS have an impact on the AARS complex formation of it.In the end, Confocal laser scanning microscope(CLSM) was used to study the subcellular location of QRS-WT and mutants, The results proved that the mutations of QRS-Lys394 and QRS-Tyr491 can promote the release from QRS complex, whereas the effect of QRS-Ser70 mutation is not obvious. This further certify the C terminal catalytic domain plays an important role in the stability of QRS complex formation. This lays a foundation for further research on tumor growth inhibition.