Stimulation of T and B cells by various agents is followed by expression of the GM-CSF gene in T cells. In T cells, we have shown that the expression of this gene in these cells is post-transcriptionally controlled mainly by stabilization of the mRNA. The mechanism of mRNA degradation and stabilization is being examined using murine EL-4 thymoma cells as a model system for T cells, and murine M12.4.1 lymphoma cells as a model system for B cells. Induction of GM-CSF mRNA in EL-4 cells by the Ca++-ionoiphore A23187 occurs by stabilization of the mature transcript in the cytoplasm. Using CAT constructs in which portions of the GM-CSF 3 prime UTR have been substituted for the SV40 poly adenilation signals and transfection of EL-4 cells, we have determined which sequences in the GM-CSF mRNA regulate degradation and stabilization. The AU-rich elements in the 3 prime UTR are principally responsible for mRNA degradation. mRNA stabilization is mediated by two regions within the GM-CSF mRNA half-life. Using PCR, we have produced linker-substitution mutations of the GM-CSF 3 prime UTR and have mapped the A23187-responsive elements of the 3 prime UTR to the AU-rich boxes and to a 60 bp region upstream from the AU-rich area. The 60 bp region is identical to the phorbol ester TPA-responsive element. Using PCR analysis of nuclear RNA with probes specific for GM-CSF intron sequences, we found that induction of GM-CSF mRNA in M12.4.1 cells by IL-1 occurs by stabilization of the nuclear precursor RNA followed by stabilization of the mature transcript in the cytoplasm. IL-4 inhibits the IL-1 induced production of GM-CSF in M12.4.1 cells. This inhibition is not regulated by changes in the transcription rate of GM-CSF gene. IL-4 causes only a slight decrease in the half-life of the mature transcript in the cytoplasm. The inhibitory effect of IL-4 is mainly due to intranuclear destabilization of the primary transcripts of GM-CSF.
