Investigations on mechanisms of transcription regulation have been the main focus in gene expression over the last two decades. It is increasingly apparent, however, that mRNA degradation is an important aspect of gene expression and its regulation. Toxicants can perturb gene expression at the level of transcriptional initiation through regulatory trans-acting factor(s) at specific cis-acting elements such as ARE, EpRE, or XRE. The steady-state level of a mRNA is established by an equilibrium between its relative rates of synthesis and degradation. There has been little mechanistic investigation on the effects of xenobiotics on mRNA stability. The pentobarbital (PB)-inducible Drosophila glutathione S-transferase (GST) D gene family offers a unique opportunity for such an investigation. The gstD1 gene is regulated by PB at the level of transcription initiation whereas regulation of gstD21 mRNA occurred mainly at the level of mRNA stability. The increased gstD1 mRNA led to an increased GST D1 protein level. By contrast a 20-fold increase in gstD21 mRNA by PB treatment did not change the GST D21 protein level. Since GST D21 is a relatively stable protein, these findings led to an hypothesis that a trans-acting factor (or factors) induced or activated by PB may be responsible for the increased stability of gstD21 mRNA through specific interactions. Such interactions, which protect gstD21 mRNA from degradation, also inhibit its translation. Therefore, there was no increase in GST D21 protein level despite a 20-fold increase of the mRNAs. This proposal intends to test this hypothesis and to delineate a molecular outline of the mechanism(s) regulating gstD21 mRNAs in the presence of PB.
The specific aims are: (1) To define the gstD21 transcript(s) by mapping the transcription start and poly(A) addition sites and constructing a full- length cDNA clone; (2) To identify a target site(s) or cis- acting element(s) which confer stability/instability to gstD21 mRNA in vitro by gel shift analyses and nuclease protection assays; (3) To investigate the biological activity of the cis-acting element(s) in transgenic flies by using chimeric gene constructs with a reporter gene; (4) To identify and purify trans-acting factor(s) which confer(s) stability/instability to gstD21 mRNA by a combination of gel shift assay and in vitro translation assay; (5) To investigate the tissue-specific expression pattern of gstD21. The long range goals are to understand the molecular mechanisms by which xenobiotics (e.g., PB) influence gene expression at the level of RNA stability and the biological/detoxification functions of GST D21. Results from this study should enhance the overall understanding of the effects of xenobiotics in modulating mammalian and human gene activities, especially those encoding the xenobiotic metabolizing enzymes.
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