Stability changes in pim-1 proto-oncogene mRNA after mitogen stimulation of normal lymphocytes.
mRNA expression of the serine/threonine kinase protooncogene Pim-1 was investigated in mitogen-treated normal bovine lymphocytes. After stimulation with Con A and phorbol ester (PMA), normal bovine PBMC exhibited a 3.5-fold induction of pim-1 mRNA within 4 h of stimulation. By 17 h poststimulation, however, the level of pim-1 mRNA had fallen to approximately 50%. Similar transient kinetics of pim-1 expression were also observed in mitogen-stimulated bovine lymph node lymphocytes and the amount of pim-1 mRNA induced was dependent on the type of mitogen stimulus. Typically, stimulation with Con A and PMA together acted synergistically resulting in a greatly increased amount of pim-1 mRNA induction compared to stimulation with either Con A or PMA alone. To determine if an increase in pim-1 mRNA stability contributed to the overall increase in pim-1 mRNA levels observed after mitogen stimulation, RNA half-life studies were performed. At 4 h poststimulation, pim-1 mRNA in bovine lymph node lymphocytes was relatively stable with a t1/2 of approximately 80 min; at 17 h poststimulation, however, the t1/2 (approximately 35 min) was markedly decreased. In addition, cyclohexamide treatment was found to markedly increase the stability of pim-1 transcripts in bovine PBMC suggesting that a protein synthesis-dependent posttranscriptional pim-1 mRNA degradation pathway may be involved in the regulation of pim-1 mRNA levels in lymphoid cells. To investigate the possible contribution of the destabilizing (UAUU)n motif to pim-1 mRNA stability, the t1/2 of the short 2.4-kb germ cell-specific pim-1 transcript found in rat testes was compared to that of the longer 2.8-kb somatic transcript expressed in stimulated rat lymphocytes. t1/2 determination experiments showed that the 2.4-kb testes-specific transcript, which is missing the destabilizing (UAUU)n sequence, was quite stable (t1/2 much greater than 6 h). In contrast, the t1/2 of the longer 2.8 kb somatic cell pim-1 transcript that contains this A/U-rich sequence motif was found to be much shorter (t1/2 approximately 130 min) in mitogen-activated rat lymphocytes. Together these findings indicate that the transient induction of pim-1 gene expression is associated with normal lymphocyte activation and that the stability of pim-1 transcripts is regulated in lymphocytes during the course of activation. In addition, the differences in pim-1 mRNA stability observed in germ cells and lymphocytes of the same animal are likely the result of molecular mechanisms involving differential regulation by the message destabilizing (UAUU)n motif.
Wingett
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D
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164.
Reeves
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R
Magnuson
N S
NS
D000818:Animals; D002417:Cattle; D003208:Concanavalin A / Q000494:pharmacology; D005786:Gene Expression Regulation; D016259:Genes, myc; D066298:In Vitro Techniques; D008198:Lymph Nodes / Q000166:cytology; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D051573:Proto-Oncogene Proteins c-pim-1; D011519:Proto-Oncogenes; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D013154:Spleen / Q000166:cytology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013950:Thymus Gland / Q000166:cytology; D013997:Time Factors
D011518:Proto-Oncogene Proteins; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D003208:Concanavalin A; C496637:Pim1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051573:Proto-Oncogene Proteins c-pim-1; D013755:Tetradecanoylphorbol Acetate