Nuclear RNA polymerase activities and poly(A)-containing mRNA accumulation in cultured AKR mouse embryo cells stimulated to proliferate

Abstract

Summary When non-growing AKR-2B mouse embryo cells are stimulated to proliferate by changing from serum-deficient to fresh media containing 10% serum, there is a consistent lag of 12 h before the onset of DNA synthesis. Endogenous DNA-dependent RNA polymerase activities, binding and initiation sites on isolated chromatin for exogenous RNA polymerase, and the rate of accumulation of poly(A)-containing polysomal RNA has been examined in AKR-2B cells with emphasis on the interval between stimulation and the onset of DNA synthesis. RNA polymerase type II activity, which is responsible for transcription of heterogeneous nuclear RNA (hnRNA), was increased by 1 h after stimulation and at 6 h reached peak levels which were 60–100% greater than the activity in resting cells. A rifampicin challenge method to assay for E. coli RNA polymerase binding and initiation sites on isolated chromatin was used to assay for changes in the amount of DNA available as a template for transcription. The results of this assay showed a slight decrease in the number of binding and initiation sites at 4 h followed by a slight increase at 6 h. The rate of accumulation of poly(A)-containing mRNA in polysomes showed a pattern and magnitude of increase following stimulation which was considerably different from that of RNA polymerase type II activity. There was a 4.5-fold increase over the resting levels by 2 h following stimulation. This enhanced level was maintained at all time points examined prior to the onset of DNA synthesis. These data suggest that both transcriptional and post-transcriptional mechanisms are responsible for the marked increase in polysomal poly(A)-containing mRNA observed after resting cells are stimulated to proliferate.