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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK050080-05
Application #
6041264
Study Section
Biochemical Endocrinology Study Section (BCE)
Program Officer
Margolis, Ronald N
Project Start
1995-08-01
Project End
2004-11-30
Budget Start
2000-01-01
Budget End
2000-11-30
Support Year
5
Fiscal Year
2000
Total Cost
$214,291
Indirect Cost
Name
University of Illinois Urbana-Champaign
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820
Mao, Chengjian; Flavin, Kathryn Goolsby; Wang, Stanley et al. (2006) Analysis of RNA-protein interactions by a microplate-based fluorescence anisotropy assay. Anal Biochem 350:222-32
Wang, Stanley Y; Ahn, Bonnie S; Harris, Rebecca et al. (2004) Fluorescence anisotropy microplate assay for analysis of steroid receptor-DNA interactions. Biotechniques 37:807-8, 810-7
Dodson, Robin E; Goolsby, Kathryn M; Acena-Nagel, Maria et al. (2003) RNA gel shift assays for analysis of hormone control of mRNA stability. Methods Enzymol 364:350-61
Goolsby, Kathryn M; Shapiro, David J (2003) RNAi-mediated depletion of the 15 KH domain protein, vigilin, induces death of dividing and non-dividing human cells but does not initially inhibit protein synthesis. Nucleic Acids Res 31:5644-53
Dodson, Robin E; Shapiro, David J (2002) Regulation of pathways of mRNA destabilization and stabilization. Prog Nucleic Acid Res Mol Biol 72:129-64
Kanamori, H; Shapiro, D J (1999) Differential display combined with a regulated transient expression system. Biotechniques 26:1018-20
Zhang, C C; Krieg, S; Shapiro, D J (1999) HMG-1 stimulates estrogen response element binding by estrogen receptor from stably transfected HeLa cells. Mol Endocrinol 13:632-43
Kanamori, H; Dodson, R E; Shapiro, D J (1998) In vitro genetic analysis of the RNA binding site of vigilin, a multi-KH-domain protein. Mol Cell Biol 18:3991-4003
Dodson, R E; Shapiro, D J (1997) Vigilin, a ubiquitous protein with 14 K homology domains, is the estrogen-inducible vitellogenin mRNA 3'-untranslated region-binding protein. J Biol Chem 272:12249-52