ИСТИНА

Translation of individual mRNAs is affected by many external stimuli including small molecule drugs used in therapy of human diseases. Under conditions of stress, eukaryotic cells trigger multiple signaling pathways to adapt their gene expression program to the changing environment. An initial response often includes reformatting the cellular translational landscape. Modifications of translational machinery components, such as eIF2 phosphorylation or eIF4F inactivation, may occur within a few minutes and last for longer periods. However, in time scale of greater then 1-2 hours, secondary effects come to the forefront, including transcription response and apoptotic programs triggered in the cases of severe stress. The translational branch of the stress response is hard to investigate by using conventional DNA reporter constructs. This is due to a large amount of proteins pre-accumulated already before the application of a stimulus, as well as many artifacts associated with this method. We have developed the fleeting mRNA transfection technique (FLERT), a method of monitoring immediate effects of stress inductors and protein synthesis inhibitors on translation of individual reporter mRNA in cultured mammalian cells. This technique narrows the time scale of analysis up to 1-2 hours and thus allows discriminating the translational regulation from other types of gene expression control. FLERT application to study the effects of oxidative stress, unfolded protein response and small molecule inhibitors on translation efficiency of several reporter mRNAs, which differ in their translation initiation mechanism, will be described. We further established a method of continuous luciferase measurement in cultured mammalian cells and applied it for studying the intracellular translation of mRNA reporter constructs. This let us to investigate how mRNA translational properties change under conditions of various cellular stresses in real time.