Using vitellogenin mRNA stabilization as a model, we are investigating the posttranscriptional regulation of mRNA stability by estrogen. This proposal focuses on our identification and cloning of vigilin as the estrogen-inducible protein which binds to a segment of the vitellogenin mRNA 3'- untranslated region important in estrogen-mediated stabilization. This intriguing, but largely unstudied, protein is conserved in all eukaryotic cells, and contains 14 distinct RNA binding domains. Vigilin forms a multi-component estrogen-regulated complex by binding to specific mRNAs, tRNA and elongation factor lalpha(EF lalpha).
The Specific Aims are: (1) To test the hypothesis that vigilin controls mRNA degradation by functioning as an integrator protein, forming a multi-component complex which links the effects of estrogen and other specific hormone signals with sensing of the overall rate of cell protein synthesis. To identify additional vigilin interaction partners we will use yeast 2-hybrid analysis, and RNA gel shifts assays. We will prepare targeted disruptions of the vigilin gene in DT40 cells and use these cells, pull-down experiments RNA gel shift assays and mammalian 2-hybrid studies carry out physical and functional mapping of the vigilin-mRNA-tRNA-EF lalpha complex. We will determine whether vigilin s interactions with tRNA and EF lalpha alter its affinity for specific mRNAs. To analyze the functional importance of vigilin-mRNA-tRNA-EF lalpha-interactions in the control of mRNA stability, we will carry out transfections in the vigilin cell line and analyze the effects of different vigilin levels and of different mRNA translation rates on mRNA degradation. We will regulate translation rates and vigilin levels in the reticulocyte lysate protein-synthesizing system and examine the degradation of mRNA targets by the polysomal mRNA endonuclease, PMR-1. (2) To investigate how estrogen and changes in cell growth and protein production induce vigilin, we will clone and analyze the vigilin promoter and analyze the regulation of vigilin transcription by estrogen and by growth factors. We will evaluate the potential role of autoregulation by free vigilin in control of vigilin levels. (3) To evaluate the role of the PMR-1 mRNase in vitellogenin mRNA degradation, we will identify the initial vitellogenin mRNA cleavage site and analyze the effect of PMR-1 on vigilin-controlled mRNA degradation in cell-free systems and in the vigilin cell line. These studies should provide new insights into a novel link between estrogen receptor action, protein synthesis rates, and the regulation of mRNA degradation.
