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.
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164.
D
D
Wingett
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R
Reeves
N S
NS
Magnuson
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