Abstract
Glucocorticoids regulate the expression of the G(1) progression factor, cyclin D3. Cyclin D3 messenger RNA (CcnD3 mRNA) stability decreases rapidly when murine T lymphoma cells are treated with the synthetic glucocorticoid dexamethasone. Basal stability of CcnD3 mRNA is regulated by sequences within the 3'-untranslated region (3'-UTR). RNA-protein interactions occurring within the CcnD3 3'-UTR have been analyzed by RNA electrophoretic mobility shift assay. Three sites of RNA-protein interaction have been mapped using this approach. These elements include three pyrimidine-rich domains of 25, 26, and 37 nucleotides. When the cyclin D3 3'-UTR was stably overexpressed, the endogenous CcnD3 mRNA was no longer regulated by dexamethasone. Likewise, overexpression of a 215-nucleotide transgene that contains the 26- and 37-nucleotide elements blocks glucocorticoid inhibition of CcnD3 mRNA expression. These observations suggest that the 215-nucleotide 3'-UTR element may act as a molecular decoy, competing for proteins that bind to the endogenous transcript and thereby attenuating glucocorticoid responsiveness. UV-cross-linking experiments showed that two proteins of approximate molecular weight 37,000 and 52,000 bind to this 3'-UTR element.
Highlights
Malignant lymphoid cells of thymic origin cease to proliferate and often die when exposed to glucocorticoids [1, 2], and glucocorticoids are an important tool for treatment of leukemias and other malignant and nonmalignant lymphoproliferative diseases
Cyclin D3 mRNA is very stable in P1798 cells treated with actinomycin D
We observed no decrease in CcnD3 mRNA after 24 h in the presence of actinomycin D [5], which leads us to suspect that actinomycin D affects the turnover of CcnD3 mRNA
Summary
Malignant lymphoid cells of thymic origin cease to proliferate and often die when exposed to glucocorticoids [1, 2], and glucocorticoids are an important tool for treatment of leukemias and other malignant and nonmalignant lymphoproliferative diseases. The rate of degradation of CcnD3 mRNA increases within 2 h after addition of glucocorticoids, to the extent that a 50% decrease in mRNA abundance occurs within 60 –90 min after addition of actinomycin D to glucocorticoid-treated cells [5]. This posttranscriptional mechanism of gene expression, coupled with the short half-life of the cyclin D3 protein, ensures a rapid response to glucocorticoids. The experiments described in this paper were designed to test the hypothesis that the 3Ј-UTR of CcnD3 mRNA contains specific protein-binding elements that are involved in glucocorticoid regulation of degradation of the mature transcript. This element interacts with at least two proteins, and this interaction is necessary for glucocorticoid-mediated destabilization of CcnD3 mRNA
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