Changes in ribosomal RNA processing paths in resting and phytohemagglutinin-stimulated guinea pig lymphocytes.

Abstract

Processing of rRNA was examined in resting and phytohemagglutinin-stimulated guinea pig lymphocytes. Synthesis of 1.7 (28S) and 0.7 (18S) X 106 dalton rRNA was more than 4-fold greater in phytohemagglutinin-stimulated than in resting cells. A 5-to 10-fold increase in flux of molecules through a 2.3 X 106 dalton RNA occurred without a concurrent change in the flux through a 2.6 X 106 dalton fraction in phytohemagglutinin-stimulated cells. In both resting and phytohemagglutinin-stimulated lymphocytes, the 2.3 X 106 dalton intermediate equilibrated with [3Hlmethyl label and pulse-chased prior to the 2.6 X 106 dalton RNA. The data indicate at least two processing paths in guinea pig lymphocytes; one proceeds to rRNA via a 2.3 X 106 dalton intermediate, and another proceeds via a 2.6 X 106 dalton RNA. The increase in rRNA synthesis in phytohemagglutinin-stimulated cells occurs primarily through that path containing the 2.3 X 106 dalton intermediate.In eukaryotes ribosomal RNA (rRNA) is transcribed as a large precursor molecule which is then processed through a series of intermediates into the definitive rRNA products (1, 2). Under certain conditions of metabolic transition within the cell, the rates of synthesis of both the precursors and the definitive rRNA are altered (3-6). Furthermore, data obtained in HeLa cells (7,8), regenerating liver and lens cells (9, 10), hormone-stimulated uterine and mammary gland cells (11,12), and human peripheral lymphocytes (13) indicate that control of rRNA production may not reside exclusively in the rate of transcription of the initial precursor molecule. The processing of this fraction could add a posttranscriptional mechanism for regulating quantitative or qualitative production of rRNA.Phytohemagglutinin (PHA)-induced transformation of lymphocytes provides a system in which gross alterations in the metabolic state of the cell are readily induced. The cells undergo a marked change in morphology and in biochemical metabolism after treatment with PHA, and one of the earliest events is a stimulation of the synthesis and processing of rRNA (14,15).In this paper data are presented which suggest that in the guinea pig lymphocyte the 1.7 X 106 dalton (1.7 MDal) (28S) and 0.7 MDal (18S) rRNA are processed from a 4.4 MDal (45S) precursor through one of at least two processing pathways and that one effect'of PHA is to stimulate processing through only one of these paths. A preliminary report has been communicated (16). METHODSIsolation and Cultures of Lymphocytes. Cervical, axillary, and mesenteric lymph nodes from male guinea pigs were trimmed of fat and teased apart in 10 ml/g wet weight The cells were then suspended at a final concentration of 3.6 or 5.0 X 106 cells per ml in modified medium supplemented with 20 ,og/ml of glutamine, 100 units/ml of penicillin, 100 jig/ml of streptomycin, and 10% heat-inactivated fetal calf serum (complete modified medium). Stationary cultures were maintained at 370 and pH 7.2 in a humidified atmosphere of 5% C02-95% air in 10 X 12 mm round-bott...

Regulation of ribosomal ribonucleic acid levels in growing, 3H-arrested, and crisis-phase WI-38 human diploid fibroblasts

“…As for cultured cells, in fusing quail myoblasts, for example, transcription continues at the same rate as in growing cells but the turnover of rRNA increases to limit the rRNA content of the cells (Bowman & Emerson, 1977). In growth-arrested mouse 3T3 cells and chicken fibroblasts, the rate of rRNA turnover also increases sharply (Abelson et al, 1974;Weber, 1972), but in mouse fibroblast lines (Johnson et al, 1976) and resting lymphocytes (Cooper, 1973; Purtell & Anthony, 1975), qualitative changes in rRNA synthesis and in the efficiency of processing have also been suggested. The present study had two aims: to attempt a more quantitative assessment of every step in RNA metabolism in a model case and to analyze a number of growth states for a single cell type.…”

Conformational changes in deoxyribonucleic acid during transcription

“…The concentration of DNA was determined by the modified diphenylamine method of Burton (1956). Whole-cell guinea pig RNA, a gift from A. Berkman, was isolated according to the procedure of Cooper & Kay (1969) as modified by Purtell & Anthony (1975). RNA complementary to calf thymus DNA was synthesized in vitro in a reaction mixture containing 1 mg of calf thymus DNA and 200 units of £.…”

Growth Factors and Regulation of Cell Growth

“…The intracellular concentrations of amino acids in quiescent cells are higher than in rapidly growing cells; hence it is unlikely that, under normal circumstances, in creased amino acids per se stimulate cell proliferation (134). Transcription, RNA processing, the assembly of ribosomal subunits into polysomes, initia tion and elongation of polypeptide chains are all stimulated after cells are treated with growth factors (135)(136)(137). New ribosomes are usually made and, apparently, new species of transfer RNA as well (135).…”