Polysomes but not translation are required for estradiol-mediated stabilization of Xenopus laevis vitellogenin mRNA
Blume, John Eric
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Permalink
https://hdl.handle.net/2142/19362
Description
Title
Polysomes but not translation are required for estradiol-mediated stabilization of Xenopus laevis vitellogenin mRNA
Author(s)
Blume, John Eric
Issue Date
1989
Doctoral Committee Chair(s)
Shapiro, David J.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
The role of translation in the estrogen mediated stabilization of Xenopus laevis vitellogenin mRNA was examined. Using a method developed for total liver polysome preparation, the efficiency of translation of vitellogenin mRNA was determined by examining the distribution of the vitellogenin polysomes within total polysomes during estrogen induction, when the message is stabilized, and during withdrawal, when the message is destabilized. The vitellogenin mRNA was loaded with a full complement of ribosomes in both cases. This indicates that translational efficiency does not change upon destabilization of the message. To further the potential requirement for translation, the dose response curves and effects for two protein synthesis inhibitors, 2-(4-Methyl-2,6-dintroanilino)-N-methylpropionamide (MDMP) and cycloheximide (CHX), were determined. MDMP, while inhibiting 89% of translation, caused mRNA's to lose their complement of ribosomes. CHX, while inhibiting 96% of translation, caused mRNA's to retain a full complement of non-translating ribosomes. When vitellogenin mRNA message stability in the presence of estradiol-17$\beta$ and these inhibitors was measured, the requirement for ribosomal loading for stabilization by estrogen to occur became apparent. Vitellogenin mRNA in the presence of hormone and CHX was stable; vitellogenin mRNA in the presence of hormone and MDMP decayed with a half-life similar to that observed during hormone withdrawal. There is no change in the association of vitellogenin mRNA with the membrane-bound polysome fraction during induction, withdrawal, or during treatment with the inhibitors in the presence of hormone. To determine if ribosomes may be required to be in close proximity to a sequence and/or secondary structure at the 3$\sp\prime$ end of the vitellogenin mRNA, the four vitellogenin 3$\sp\prime$-untranslated sequences were isolated and sequenced. Sequence comparison reveals a region conserved in both content and position among the four vitellogenin genes; Py-AATGTPuPy-TT. This sequence is also found in three other genes whose mRNA is regulated by estrogen at the level of message stability. A potential role for the requirement for a full ribosome complement and the conserved sequence motif is discussed, as is a general model for the regulation of mRNA stability.
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