Primary T-cells are metabolically quiescent, with little DNA, RNA or protein synthesis. We are studying the role of eIF-2 and elF-4E expression in the mechanism of translational activation upon mitogenic stimulation. mRNA levels of eIF-2 alpha, eIF-2 beta and elF-4E all increase greatly in the 1st 24 hour of stimulation. Nuclear run-on analysis shows that the increase in eIF-2 alpha mRNA is not due to a significant increase in the relative level of transcriptional initiation, nor is it due to an increase in mRNA half-life. Dot-blot analysis of nuclear RNA shows that eIF-2 alpha hnRNA increases dramatically, using either intron or exon-specific probe. This suggests that the increase in eIF-2 alpha hnRNA is regulated at a very early point, but subsequent to transcriptional initiation. We are currently attempting to block the increase of eIF-2 alpha with antisense DNA to see if the 50 fold increase is necessary for translational activation. With activation there is also a dramatic shift of mRNA and ribosomes to polysomes indicating efficient translation. In spite of the large increase in mRNAs, by 24 hours the number of eIF-2 alpha, eIF-2 beta and elF-4E molecules per cell only increase 2-3-fold, similar to the increase in ribosome number per cell. This suggests a mechanism involving modification of pre-existing factors. We have already shown that with T-cell activation, the phosphorylation of elF-4E, but not elF-2 alpha or eIF-2 beta increases. Phosphorylation of elF-4E has been shown by other workers to correlate with increases in translation activity. Our work will continue to evaluate the participation of these and other factors, as well as study the modification of pre-existing factors to determine how their activity is influenced by activation, ultimately contributing to the increase in protein synthesis.
