Dihydrofolate reductase gene expression in cultured mouse cells is regulated by transcript stabilization in the nucleus

Abstract

Methotrexate-resistant cells, which contain a 500-fold amplification of dihydrofolate reductase (DHFR) genes, were used as a model system for studying the regulation of DHFR gene expression during growth stimulation. We have shown that a threefold increase in DHFR mRNA levels following growth stimulation results from a corresponding increase in DHFR mRNA production (i.e., delivery to the cytoplasm) and is not the result of a change in DHFR mRNA half-life. We previously showed that the increase in DHFR mRNA production during growth stimulation is not accompanied by an increase in the relative rate of transcription of the DHFR gene. This suggested that changes in DHFR mRNA production during growth stimulation are due to changes in the stability of DHFR transcripts in the nucleus. Using continuous labeling experiments in vivo comparing the stability of DHFR RNA with specific reference sequences, we show that in growing cells most DHFR transcripts were converted to mRNA, whereas in resting cells the majority of DHFR transcripts were rapidly degraded in the nucleus. There was no significant difference in the rate of processing and transport of stable DHFR transcripts. Therefore, changes in the stability of DHFR RNA in the nucleus control the amount of mRNA available for translation in the cytoplasm.