Nucleic Acids Research, 1993, Vol. 21, No. 18 4356-4362
© 1993
MOLECULAR BIOLOGY |
rseB, a chromosomal locus that affects the stability of a temperature-specific surface protein mRNA in Tetrahymeana thermaophil
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences 4301 W. Markham, Little Rock AR 72205, USA
*To whom correspondence should be addressed
Received March 29, 1993. Revised July 27, 1993. Accepted July 27, 1993.
In Tetrahymena thermophila, the expression of a temperature-specific surface protein known as SerH3 Is primarily controlled by a temperature-dependent change In the stability of the mRNA that encodes this protein. At 30°C the SerH3 mRNA displays a half-life of 60 minutes while at 40°C the half-life decreases to only 3 minutes. We used a Tetrahymena mutant cell line (rseB) defective In expression of SerH3 at 30°C to explore the mechanisms involved in temperaturedependent mRNA stability. The results of In vitro nuclear run-off assays and Northern and slot blot analysis of cytoplasmlc and nuclear RNAs show that the rseB locus encodes a temperature-sensitive product that has no effect on SerH3 gene transcription or the steady-state levels of SerH3 nuclear RNA. However, the product of this locus does have a dramatic effect on cytoplasmic levels of the SerH3 mRNA at 30°C, Indicating that SerH3 gene expression Is affected post-transcrlptlonally within the cytoplasm. To explore the possibility that the rseB locus controls SerH3 mRNA stability we developed an In vitro mRNA decay assay. This assay successfully duplicates the differential decay of the SerH3 mRNA observed In wildtype cells grown at different temperatures. The apparent half-life of the SerH3 mRNA in cytoplasmlc extracts derived from cells grown at 30°C Is approximately 45 minutes while In cytoplasmlc extracts derived from cells grown at 40°C it is only 6 minutes. When similar experiments are performed using extracts prepared from the Tetrahymena rseB cell line, we find that the SerH3 mRNA Is only stable In extract prepared from cells grown under conditions in which the mRNA accumulates to detectable levels in the cytoplasm. These results Indicate that the product of the rseB locus Is a frans-acting cytoplasmlc factor that exerts its effect on SerH3 gene expression by regulating SerH3 mRNA stability.
+Present address: La Jolia cancer Research Foundation, 10901 N. Torrey Pines Road, La Jolua, CA 92037, USA
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