Estrogen administration to male Xenopus laevis causes the post- transcriptional suppression of serum albumin mRNA via an estrogen receptor-dependent mechanism that occurs within hours after the transcriptional induction of the genes for the yolk protein precursor vitellogenin.
The specific aims of this proposal each address a fundamental question about post-transcriptional regulation by estrogen: 1) To determine if albumin mRNA is selectively degraded in the nucleus or the cytoplasm, and if this is nuclear to examine the role of polyadenylation and/or RNA splicing in this process; 2) To examine changes in both steady- state levels and turnover of albumin mRNA in cultured hepatocytes to further define the role of the estrogen receptor and to determine whether the suppression of albumin mRNA requires the synthesis of a new gene product; 3) To investigate coordinate post-transcriptional regulation of serum protein mRNAs in order to identify a consensus sequence or secondary structure that defines target specificity. The long range goal is to identify the molecules and mechanisms involved in estrogen- dependent post-transcriptional regulation of gene expression. Estrogen-induced changes in albumin nuclear precursor RNA will examined initially by Northern blot analysis using as probes the cloned cDNA and a cloned intron from the albumin gene. Quantitative changes in nuclear precusor RNA will then be determined by S1 protection experiments. The role of polyadenylation will be examined by chromatography of nuclear precursor RNA on poly(U)-cellulose and by direct analysis of poly(A) length on albumin mRNA at intervals after estrogen administration. Subcloned fragments of either pure exon sequence or spanning intron/exon boundaries will be used in S1 protection experiments to determine if estrogen might cause alterations in albumin mRNA splicing. Detailed studies will be performed on albumin mRNA turnover in cultured hepatocytes and inhibitors of protein and RNA synthesis will be used to determine if new gene products are required to destabilize albumin mRNA. Finally, if the data indicate a cytoplasmic mechanism the focus will shift to coordinate regulation of serum protein mRNAs to search for common features that may play a role in the targeting of mRNA for changes in stability. Future studies will utilize DNA-mediated gene transfer into cultured hepatocytes as a functional assay for any specific sequences identified as components in the regulation of albumin gene expression.
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