We have identified an estrogen-inducible protein which binds with high specificity to a segment of the 3'-untranslated region of the estrogen- stabilized mRNA encoding the egg yolk precursor protein, vitellogenin. Since this protein is widely distributed in different tissues of Xenopus laevis, is regulated by both estrogen and testosterone, and we have identified a human homologue, it likely plays an important role in steroid hormone control of mRNA degradation. Our approach is to clone, express and characterize the protein, to investigate its regulation by estrogen and other hormones, determine the precise sequence and structure of the mRNA binding site, identify additional mRNAs which bind the protein and investigate its role in the regulation of mRNA stability. The high specificity with which the protein binds to its recognition sequence, and our development of a nitrocellulose filter binding assay make direct screening of a cDNA expression library our preferred method for cloning and initial expression. If this proves impossible, we will use biochemical techniques including RNA affinity chromatography to purify the protein, obtain partial amino acid sequence, and use degenerate oligonucleotides to screen a cDNA library. The Xenopus cDNA clone will be used to isolate a human binding protein cDNA clone. Nucleic acid probes and antibodies to the expressed protein will be used to determine whether estrogen induces the mRNA and protein, or increases the affinity of pre-existing protein for its mRNA binding site. We will then carry out a more detailed analysis of the regulatory mechanism. To further characterize the mRNA binding site we will use an iterative in vitro selection method, RNase protection and mutagenesis. The in vitro selection provides an unusual approach to identifying a consensus mRNA binding site, and generating up-binding mutants of the binding site, and will facilitate modeling the secondary structure of the binding site. We will use the sequence and structure of the consensus binding site to identify other mRNAs whose stability is regulated which contain potential binding sites for the protein. We will determine the relationship between estrogen induction, binding of the protein to its recognition sequence and control of vitellogenin mRNA stability. We will also examine the role of the binding protein in controlling the stability of one human mRNA in which we identify a binding site. We will determine whether the initial RNase cleavage site is located near the protein binding site. These studies should provide important insights into steroid hormone control of mRNA stability.
