Transport of RNA from nucleus to cytoplasm following mitogenic stimulation of human lymphocytes

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Abstract
An improved method affording clean separation of nuclei from cytoplasm was utilized to study the kinetics of appearance of [3H]uridine-labeled polyadenylated (poly(A)+) and nonpolyadenylated (poly(A)-) RNA in the cytoplasm of human lymphocytes stimulated with the mitogen concanavalin (Con)A. Studies utilizing lymphocyte preparations purified on Ficoll-Hypaque gradients showed that the earliest observable increase in labeled total cell RNA occurred after 12 h of exposure to Con A. Highly significant increases in levels of uridine-labeled poly(A)- RNA were detected in the cytoplasm of lymphocytes which were exposed to mitogen for 6 h; these cells were pulsed with [3H]uridine for 2 h prior to harvest. The early effects of mitogen may be to increase processing and transport of RNA rather than to increase the production of gene transcripts. At least 13 h of exposure to Con A was required before labeled poly(A)+ RNA appeared in the cytoplasmic extracts of stimulated cells, and it did not appear in those of resting cells. The lag between addition of isotope and appearance of labeled RNA in the cytoplasm was inversely related to the duration of incubation of the cells with mitogen. Once the label appeared in the cytoplasm, its rate of appearance and its final level were similar at 12 and 36 h of incubation with Con A. Mitogen apparently acts mainly to accelerate the processing and transport of RNA at least during the first 12 h of mitogenic stimulation. A concentration (0.05 .mu.g/ml) of actinomycin D (which is known to inhibit specifically the transcription of nucleolar RNA in a variety of cell types) affects the transcription of poly(A)+ RNA in human lymphocytes; in some experiments, specific inhibition of nucleolar RNA synthesis was observed at 0.005 .mu.g actinomycin D/ml.