Increased efficiency of translation of ornithine decarboxylase mRNA in mitogen‐activated lymphocytes

Abstract

Ornithine decarboxylase (ODC) mRNA was elevated ninefold by 6 h following concanavalin A (ConA) stimulation of bovine lymphocytes. Comparison of the increases in ODC mRNA and ODC activity revealed a fivefold discrepancy, which is consistent with a change in efficiency of translation of ODC mRNA. In resting cells, 45% of the total ODC mRNA was associated with particles sedimenting at about 40 S, and therefore was not translated. The untranslated ODC mRNA in resting cells could be completely shifted into polysomes by a 15-min treatment of the cells with appropriate concentrations of cycloheximide. In activated cells, the proportion of ODC mRNA in untranslated material was reduced to 18%. This shift in distribution of ODC mRNA occurred between 6 h and 12 h following mitogen stimulation with no increase in the cellular level of this message. The rate of synthesis of ODC protein was found in increase twofold between 6 h and 12 h, paralleling the increase in the amount of ODC mRNA associated with polysomes. Thus, in this time frame, a decrease in the amount of untranslated ODC mRNA with a corresponding increase in the amount associated with polysomes leads to an increase in the biosynthesis of ODC with no change in the cellular level of the message. These changes in translational efficiency were not observed with actin mRNA.Increased rates of synthesis of the polyamines, putrescine, spermidine and spermine, accompany a variety of cellular stimuli, including responses to growth factors, tumor promoters and hormones [I, 21. These increases are required for cell proliferation [2-41. The first step of polyamine biosynthesis is catalyzed by ornithine decarboxylase (ODC), which is also a major site of regulation of the pathway. The cellular levels of ODC activity and protein are elevated rapidly in response to stimuli. The rapid kinetic course of ODC induction is due, at least in part, to the rapid turnover of this protein. Recently, cloning of cDNAs coding for ODC has facilitated analysis of the behavior of ODC mRNA [5-81. Elevated levels of ODC mRNA accompany induction of the enzyme in at least three systems: serum-stimulated mouse fibroblasts [7], kidneys from androgen-treated mice [6 -81 and rat pheochromocytoma cells treated with nerve growth factor [9]. In this paper we show that induction of ODC during lymphocyte mitogenesis results from both increases in the level of ODC mRNA and in the efficiency of its translation. This latter mode of control is due to an increase in the fraction of ODC mRNA associated with polysomes, which occurs after the elevation of ODC mRNA level. MATERIALS AND METHODS Cell cultureBovine lymphocytes were purified from suprapharyngeal lymph nodes as previously described [lo]. Cells were stimulat- RNA purification, electrophoresis and hybridizationTotal RNA was purified from cells at various times after ConA addition by the guanidium isothiocyanate method of Chirgwin et al. [ll]. RNA from sucrose gradients was prepared by incubating each gradient fraction with SDS and proteinase K (final conce...

Cell adhesion regulates gene expression at translational checkpoints in human myeloid leukocytes

“…5c). In contrast, the levels of actin were similar in eIF4E-overexpressing and wild-type cells, consistent with a previous report that its synthesis is independent of translational control (43). In addition, IL-8 was not increased in lysates or supernates of the overexpressing cells (not shown), consistent with differential regulation of UPAR and IL-8 synthesis in monocytes and with translational regulation of UPAR (Fig.…”

Selenoprotein P Expression, Purification, and Immunochemical Characterization

“…DEAE-dextran was prepared in phosphate-buffered saline supplemented with 0.1 mM chloroquine. Media were changed to DMEM without serum at the time of addition of 75 Se as above, and labeling was continued for 24 h. Polysome Fractionation and RNA Analysis-Polysome preparation from transiently transfected HEK 293 cells was carried out according to a modification of the protocol described by White et al (23). Cells were harvested as above, except that cycloheximide was added to 100 g/ml 20 min before harvest.…”

Ras Transformation of RIE-1 Cells Activates Cap-Independent Translation of Ornithine Decarboxylase: Regulation by the Raf/MEK/ERK and Phosphatidylinositol 3-Kinase Pathways

“…Ras activation in response to growth factors or constitutively active Ras mutants induces ODC activity by a complex mechanism involving transcription, translation, and changes in protein stability (8,11,12). Previous studies suggest that ODC synthesis is regulated predominantly by translation, especially at the level of initiation (13,14).…”