Introduction
The level of an mRNA in the cytoplasm represents a balance between the rate at which the mRNA precursor is synthesized in the nucleus and the rates of nuclear RNA processing and export and cytoplasmic mRNA degradation. Although most studies of gene expression have focused on transcription and its control, a great deal of information indicates that mRNA degradation and its regulation are major mechanisms that help govern cellular mRNA levels. mRNA degradation rates help to determine protein levels. The amount of protein that can be translated from each molecule of an mRNA is determined by the amount of time the mRNA persists in the cytoplasm. An mRNA with a long half-life will usually undergo a great many rounds of translation thereby amplifying effects based on regulation of gene transcription. For example in the presence of estrogen and estrogen receptor, vitellogenin mRNA exhibits along half life, thereby allowing production of vast amounts of the egg yolk proteins needed during reproduction. Conversely, an mRNA with a short half life changes its level rapidly in response to alterations in transcription rates. For these unstable mRNAs changes in transcription rates are therefore rapidly reflected by changes in the rate of protein production. Many growth factors, cell cycle regulators and transcription factors are only needed transiently by cells and are encoded by unstable mRNAs.
Our initial interest in the hormone control of mRNA stability came from early studies in which we showed that estrogen both increased the rate of transcription of the gene encoding the egg yolk precursor protein, vitellogenin, and specifically stabilized vitellogenin mRNA against cytoplasmic degradation. Xenopus Vitellogenin mRNA exhibits a half-life of 500 hours (about 3 weeks) when estrogen is present in the culture medium and 16 hours following removal of estrogen from the medium. We demonstrated that vitellogenin mRNA stabilization is a reversible cytoplasmic effect of estrogen. In subsequent work, using the estrogen stabilization of vitellogenin mRNA as a model system, we demonstrated that stabilization is a positive effect which requires hormone receptor complex, and that vitellogenin mRNA must be associated with ribosomes, but does not have to be undergoing translation in order to be stabilized by estrogen. Using a cell culture model we demonstrated that more than 90% of the 6,000 nucleotide vitellogenin mRNA could be deleted without blocking estrogen stabilization, and that sequences in the 3'-untranslated region were important in stabilization (Reviewed in Dodson and Shapiro, 2002). We next identified mRNA binding proteins able to bind to the vitelogenin mNA 3'-UTR. These studies enabled us to identify vigilin as an estrogen-inducible proein that binds to a segment of the vitelogenin mRNA 3'-untranslated region important in estrogen mediated stabilization of vitellogenin mRNA (Dodson, R.E., and Shapiro, D.J. 1997 Vigilin, a Ubiquitious Protein with 14 KH Domains is the Estrogen-Inducible Vitellogenin mRNA 3'-Untranslated Region Binding Protein. J. Biol. Chem. 272: 12249-12252). This data led to our hypothesis that estrogen induction of vigilin leads to binding of vigilin to the vitellogenin mRNA 3'-untranslated region (UTR), and that the bound vigilin protects a nuclease sensitive cleavage site in the 3'-UTR, and thereby blocks vitellogenin mRNA degradation. (A figure outlining this model is at the front of this web site) In a collaborative study with the laboratory of Prof. Dan Schoenberg, purified recombinant vigilin was shown to protect this region of vitellogenin mRNA from in vitro degradation by the sequence-selective mRNA endonuclease, PMR-1, previously shown to be important in the estrogen-mediated degradation of Xenopus albumin mRNA (Cunningham et al., 2000).
An RNA gel mobility shift assay showing that estrogen induces vigilin binding to a labeled 90 nucleotide probe containing the key segment of the vitellogenin mRNA 3'-UTR. (for methodological details see Dodson and Shapiro 2003)
Our subsequent studies on the regulation of vigilin synthesis, and on the binding of vigilin to mRNAs led us to view vigilin as an important protein in mRNA metabolism (Kanamori, H., Dodson, R.E. and 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). Our initial studies of the functions of vigilin are detailed in the next section.
Measuring mRNA degradation rates has usually been accomplished by inhibiting the synthesis of all cell mRNAs with actinomycin D, or DRB, and measuring the rate at which the mRNA of interest disappears. Although it is well known that actinomycin D and other global inhibitors of mRNA synthesis interfere with diverse cell processes and often produce artifacts in measuring mRNA degradation rates, the absence of alternatives methods has led to their continued and widespread use. To develop an improved method for measuring mRNA degradation rates we extended observations we made on the use a tetracycline-regulated promoter system (Kanamori and Shapiro, (1999) Differential Display Combined with a Regulated Transient Expression System. BioTechniques 26: 1018-1020). The system we developed involves the use of a regulated promoter to synthesize only the mRNA of interest as a pulse. The degradation of the mRNA is measured by quantitative RT-PCR and is made possible by improved methods for eliminating the transfected DNA from the RNA samples (Mao and Shapiro, unpublished). Using this technology and other techniques in vivo analyses of estrogen-regulated mRNA degradation are under way.
Some Recent Publications
Cunningham, K., Dodson, R.E., Nagel., M.A., Shapiro, D.J. and Schoenberg. D.R. (2000) Vigilin Binding Selectively Inhibits Cleavage of the Vitellogenin mRNA 3'-Untranslated Region by the mRNA Endonuclease Polysomal Ribonuclease 1. Proc. Natl. Acad. Sci. USA 97: 12498-12502.
Dodson, R.E. and Shapiro D.J. (2002) Regulation of Pathways of mRNA Destabilization and Stabilization. In K. Moldave Ed. Progress in Nucleic Acids Res. and Molec. Biol. 72: 129-164
Dodson, R.E. Goolsby, K.M., Acena-Nagel, M., Mao, C. and Shapiro, D.J. (2003) RNA Gel Shift Assays for Analysis of Hormone Control of mRNA Stability. In D.W. Russell and D.W. Mangelsdorf Eds. Methods in Enzymology: Nuclear Receptors. 364: 350-361, In Press.
Last Updated: 08/12/03