Evidence for the requirement of protein synthesis and protein kinase activity in the temperature regulated stability of a Tetrahymena surface protein mRNA

Abstract

In Tetrahymena thermophila, the expression of the temperature-specific surface protein SerH3 is controlled primarily by a temperature-dependent change in the stability of its mRNA. The change in SerH3 mRNA stability occurs very rapidly after a shift in incubation temperature. This change in temperature could affect SerH3 mRNA stability directly by producing structural changes in the mRNA or regulatory factors acting on SerH3 mRNA. Alternatively, the temperature change could act indirectly through a signal transduction pathway leading to de novo synthesis of new regulatory factors or modifications of existing regulatory factors. To address these issues, we monitored the effect of temperature on an in vitro SerH3 mRNA decay assay and the in vivo effects of a variety of inhibitors against protein synthesis and protein kinases on SerH3 mRNA stability. The results of Northern analysis of SerH3 mRNAs in an in vitro mRNA decay assay indicate that temperature alone can not change the half-life of this mRNA. Furthermore, slot blot analysis of cytoplasmic RNAs show that protein synthesis and the action of protein kinases are not required for SerH3 mRNA turnover in cells grown at 30 degrees C. In contrast, our results indicate that the rapid decay of the SerH3 mRNA in cells grown at 30 degrees C and shifted to 40 degrees C requires a one time serine/threonine phosphorylation event which occurs at the temperature shift. In addition, the data show that a regulatory protein involved in rapid SerH3 mRNA decay must be newly and continuously synthesized following the temperature shift from 30 to 40 degrees C. These data show the complexity of temperature regulated mRNA decay and indicate that phosphorylation and protein synthesis are major factors in this process.