This proposal concerns the regulation of vitellogenin and apolipoprotein gene expression by estrogens. In the avian liver, estrogen induces the expression of genes encoding proteins that serve to transport lipids and other nutrients to the ovary for deposition in the egg yolk. Among these proteins are vitellogenin (VTG) and apolipoprotein (apo) II of very low density lipoprotein. In addition to regulating transcription, estrogen also regulates the turnover of these mRNAs in the cytoplasm. Effects of estrogen on mRNA turnover have long been known to play a major role in regulating gene expression. However, little is known about the mechanisms of mRNA turnover or how estrogen alters this process in an mRNA-specific manner. The proposed experiments are focused on apoII mRNA and are designed to elucidate these mechanisms. Project I. will define RNA elements necessary for normal mRNA turnover and estrogen-mediated destabilization. ApoII genes with specific deletions in the 3'noncoding region will be expressed in an estrogen-responsive chicken liver cell line (LMH) via transient transfection and stable transformation protocols. Project II. will identify cytosolic proteins that bind to apoII mRNA, delimit mRNA elements responsible for binding, determine cell-type and mRNA specificity, and determine the effect of estrogen on mRNA binding proteins. Project III will analyze the pattern of apoII mRNA expression, turnover, and translation in the LMH cell, a newly developed, estrogen-responsive avian cell line. ApoII mRNA turnover will be examined in the presence of estrogen and during hormone withdrawal after different periods of estrogen treatment. The influence of subcellular location on apoII mNRA degradation will be examined with an apoII gene from which the signal peptide element is deleted. Project IV will test whether the 3' noncoding regions is involved in long-range base pairing interactions. Analysis of fragments of the 3' noncoding region will delimit local secondary structure domains. Structure analysis will employ nuclease and chemical modification data as constraints for computer-assisted RNA folding. A putative stem-loop structure containing endonuclease cleavage sites will be studied by two dimensional nuclear magnetic resonance spectroscopy as an initial approach to the detailed structure of a functional domain. The ling-term goal of the proposed research is to understand how apoII mRNA degradation occurs and how this process is regulated by estrogen. The regulation of mRNA turnover is a common strategy for modulating the expression of specific genes. The proposed research will answer several key questions about these important cellular processes.
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