Abstract
Regulation of the cell cycle is orchestrated by cyclins and cyclin-dependent kinases. We have demonstrated previously that overexpression of eukaryotic translation initiation factor 4E (eIF-4E) in NIH 3T3 cells growing in 10% fetal calf serum leads to highly elevated levels of cyclin D1 protein without significant increase in cyclin D1 mRNA levels, suggesting that a post-transcriptional mechanism is involved. (Rosenwald, I. B., Lazaris-Karatzas, A., Sonenberg, N., and Schmidt, E. V. (1993) Mol. Cell. Biol. 13, 7358-7363). In the present research, we did not find any significant effect of eIF-4E on polysomal distribution of cyclin D1 mRNA. However, the total amount of cyclin D1 mRNA associated with polysomes was significantly increased by eIF-4E overexpression. Further, we determined that the levels of both cyclin D1 protein and mRNA are increased in serum-deprived cells overexpressing eIF-4E. Nuclear run-on experiments demonstrated that the rate of the cyclin D1 transcription is not down-regulated in serum-deprived cells overexpressing eIF-4E. Thus, elevated levels of eIF-4E may lead to increased transcription of the cyclin D1 gene, and this effect becomes visible when serum deprivation down-regulates the rate of cyclin D1 mRNA synthesis in control cells. However, artificial overexpression of cyclin D1 mRNA in serum-deprived cells in the absence of eIF-4E overexpression did not cause the elevation of cyclin D1 protein, and this overexpressed cyclin D1 mRNA accumulated in the nucleus, suggesting that one post-transcriptional role of eIF-4E is to transport cyclin D1 mRNA from the nucleus to cytoplasmic polysomes.
Highlights
Mitogenic stimulation leads to increased rates of protein synthesis, which is required for entry of resting cells into the cell cycle [2,3,4,5]
We have demonstrated previously that overexpression of eukaryotic translation initiation factor 4E in NIH 3T3 cells growing in 10% fetal calf serum leads to highly elevated levels of cyclin D1 protein without significant increase in cyclin D1 mRNA levels, suggesting that a post-transcriptional mechanism is involved
Increase in Cyclin D1 Protein Correlates with the Level of eukaryotic translation initiation factor 4E (eIF-4E) in Serum-deprived and -stimulated Cells—We have previously reported that the steady-state levels of cyclin D1 protein, normalized to total cellular protein, are dramatically increased in eIF-4E overexpressing cells continuously growing in the presence of 10% fetal calf serum (FCS) as determined by Western blot [1]
Summary
Mitogenic stimulation leads to increased rates of protein synthesis, which is required for entry of resting cells into the cell cycle [2,3,4,5]. One of the translation initiation factors whose levels are increased after mitogenic stimulation of resting cells is the ratelimiting mRNA cap-binding protein eukaryotic translation initiation factor 4E (eIF-4E), which may be involved in unwinding of mRNA 5Ј secondary structures, mRNA splicing, mRNA 3Ј processing, and mRNA nucleocytoplasmic transport [11,12,13]. An important role for eIF-4E in cell growth has been demonstrated in experiments in which microinjection of this translation initiation factor into quiescent NIH 3T3 cells induced them to enter the S phase [14]. We have found that the levels of cyclin D1 protein, but not pRB, are and very strongly increased in continuously growing NIH 3T3 cells overexpressing eIF-4E [1]. In the present report we describe our progress on identifying the nature of regulation of cyclin D1 gene expression by translation initiation factor 4E
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