Dr. Gelehrter's laboratory has previously demonstrated that incubation of rat hepatoma cells with cyclic nucleotides results in decreased transcription from the type 1 plasminogen activator inhibitor (PAI-1) gene as well as a 3-fold increase in the rate of PAI-1 mRNA decay. The half- life of PAI-1 mRNA in HTC cells is 4.5 hours; after incubation with cyclic nucleotides, PAI-1 mRNA decays with a half-life of 1.5 hours after a 2 hour lag period. The objective of this proposal is to establish a cell- free, in vitro mRNA degradation assay system for PAI-1 mRNA and to use this system to determine the cis-acting sequences in PAI-1 mRNA and identify the trans-acting HTC cell factors that are responsible for the cyclic nucleotide-mediated destabilization of PAI-1 mRNA in HTC cells. The in vitro system is advantageous because: (i) it allows analysis of mRNA degradation without the use of transcriptional inhibitors, (ii) it allows analysis of cytoplasmic events independently of nuclear events, (iii) it may allow for the detection of mRNA decay intermediates, (iv) it may be possible to use synthetic mutant PAI-1 mRNAs to identify nucleic acid sequences involved in both basal and cyclic nucleotidc-regulated PAI- 1 mRNA decay, and (v) it provides a facile system for identifying and characterizing the trans-acting factors regulating PAI-1 mRNA degradation. Regulation of mRNA stability is, as yet, poorly understood. To date, the best studied systems are ones in which mRNAs are stabilized in the presence of hormones. The regulation of PAI-1 mRNA by cyclic nucleotides provides a favorable experimental system in which to examine hormonally- induced mRNA destabilization. Through the proposed studies, we will be able to explore the molecular mechanisms by which cyclic nucleotides regulate PAI-1 mRNA stability. Furthermore, results from these studies will provide important information about both constitutive and hormonally regulated pathways of mRNA degradation.
| Tillmann-Bogush, M; Heaton, J H; Gelehrter, T D (1999) Cyclic nucleotide regulation of PAI-1 mRNA stability. Identification of cytosolic proteins that interact with an a-rich sequence. J Biol Chem 274:1172-9 |
| Heaton, J H; Tillmann-Bogush, M; Leff, N S et al. (1998) Cyclic nucleotide regulation of type-1 plasminogen activator-inhibitor mRNA stability in rat hepatoma cells. Identification of cis-acting sequences. J Biol Chem 273:14261-8 |
