Although Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) expression is prototypic of transcripts regulated by their half-lives, how this occurs is not yet well understood. In 1986, Show and Kamen reported an AU rich element (ARE) in the 3' untranslated region (UTR) of GM-CSF mRNA confers a short half-life on otherwise stable transcripts. However, other elements contributing to the short half-life of GM-CSF mRNA probably exist, as the removal of the ARE fails to completely stabilize the construct from which it has been deleted. We are using a genetic/molecular biological approach to identify new RNA elements, possibly in the protein coding region which contribute to the instability of GM-CSF mRNA. During the past year, we have accomplished 4 milestones along the way to answering these questions. First, we have generated the parent DNA expression constructs. They consist of the following: the beta-globin 5' UTR, followed by the beta-globin coding region and the beta-globin 3' UTR; and the analogous parental construct for the GM-CSF gene. All constructs contain the appropriate intronic sequence in order to rule out splicing effects. In addition, all constructs contain restriction sites engineered into the junctions between the untranslated and coding regions in order to allow them to be exchanged while preserving the integrity of the reading frames. Second, we have successfully transfected these expression constructs into a murine fibroblast cell line and achieved stable expression of the correctly spliced, polyadenylated mRNA transcripts of the correct sizes. Third, we have obtained data indicating that the parental beta-globin mRNA is stable in our cell line, and that the parental GM-CSF mRNA has a half-life of 40 minutes. Fourth, we have generated the following chimeric constructs: the beta-globin 5' UTR and coding region followed by the 3' UTR from the GM-CSF gene; the beta-globin coding region surrounded by the 3' and 5' UTR's of the GM-CSF gene; and the GM-CSF coding region surrounded by the 3' and 5' UTR's of the beta-globin gene. When we generate cell lines that stably express the transcripts resulting from these constructs, we will make half-life determinations in order to determine whether the GM-CSF coding region contains an mRNA destablizing element. If it does, then we plan to identify and characterize this segment of mRNA.
