Stability of human follicle-stimulating hormone receptor mRNA in stably transfected cells.
In order to assess the impact of mRNA degradation on steady state levels of follicle-stimulating hormone receptor (FSHR) mRNA and on regulation of FSHR gene expression, the stability and half-life of FSHR mRNA were determined in transfected cells expressing recombinant FSHR. Time-dependent changes in FSHR mRNA content were determined by nuclease protection-solution hybridization assay (NPA) or by qualitative reverse transcription-competitive polymerase chain reaction (RT-PCR) in cultured hFSHR-YI cells, cell lines stably transfected with a human FSHR cDNA. FSHR mRNA content remained constant during 8 h control incubations of hFSHR-Y1 cells (NPA, 2.9 +/- 0.3 micrograms/mg RNA; RT-PCR, 2.7 +/- 0.3 micrograms/mg RNA). Actinomycin D (ActD, 5 micrograms/ml) inhibited mRNA synthesis, as assessed by incorporation of [3 H]uridine into total RNA, by 90% within 1 h in hFSHR-Y1 cells. No effect of ActD on cellular morphology or viability was observed. ActD caused a time-dependent decrease in FSHR mRNA content in hFSHR-Y1 cell lines with a lag time of 1 h. There were no significant differences in the rate of FSHR mRNA degradation between the two methods of mRNA quantification. The half-life of hFSHR mRNA was 3.6 +/- 0.2 h by NPA and 3.1 +/- 0.1 h by RT-PCR. The results indicated that degradation of mRNA was an important process in maintenance of steady state expression of the FSHR gene in cells stably expressing recombinant receptor.
Zhu
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Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030.
Tian
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D000306:Adrenal Cortex Neoplasms / Q000378:metabolism / Q000473:pathology; D000818:Animals; D005260:Female; D015870:Gene Expression; D051379:Mice; D011247:Pregnancy; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011962:Receptors, FSH / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D011962:Receptors, FSH; D011994:Recombinant Proteins