BACKGROUND: Eukaryotic translation initiation factor EIF-4E is a key component in the regulation of translational efficiency of MRNAs and its increased expression may accelerate cell growth and division. EIF-4E has the potential to transform rat embryo fibroblast cells by cooperation with v-Myc or adenovirus E1A. Our previous study showed that a variety of tumor cells examined exhibited elevated levels of eIF-4E gene expression. Thus it is thought that overexpression of EIF-4E can result in aberrant growth and cell transformation. EXPERIMENTAL DESIGN: To characterize rat EIF-4E, a clone containing rat eIF-4E cDNA was isolated from a rat testis cDNA library by screening with a synthetic probe prepared by the reverse transcription-polymerase chain reaction (RT-PCR) method. Based on the conserved nucleotide sequences between mouse and human eIF-4E cDNA, two oligonucleotide primers for RT-PCR were synthesized chemically. The cloned cDNA was sequenced and used as a probe for analysis of the expression level of eIF-4E mRNA in normal, differentiated rat tissues by Northern blot analysis. In situ hybridization analysis with digoxigenin-labeled antisense eIF-4E RNA as a probe was carried out to identify the eIF-4E-expressing sites in rat tissues. In addition, to analyse the phosphorylation level of EIF-4E, the proteins were fractionated from rat tissues by affinity column chromatography followed by isoelectric focusing (IEF)-gel electrophoresis. RESULTS: The nucleotide sequence of the eIF-4E cDNA is highly conserved in human, rat, and mouse. Extraordinarily elevated expression, more than 50-fold compared with that in the adult rat prostate, of eIF-4E was observed in testicular germ cells of rats of reproductive age, which was much greater than that in any tumor cell lines examined so far. The amount of EIF-4E fractionated from the adult rat testis was approximately 10 times higher than that from the adult rat liver. At least half of the purified testicular EIF-4E proteins were phosphorylated, a ratio similar to that in other rat tissues such as liver. In situ hybridization analysis demonstrated that elevated expression of eIF-4E mRNA was mainly observed in post-meiotic germ cells. CONCLUSIONS: Full activity of EIF-4E in translation requires the phosphorylation of the protein on a specific serine residue. Thus the elevated level of EIF-4E observed in the adult rat testis should be reflected in increase of the functional activity of EIF-4E. Based on the results of in situ hybridization analysis and characterization of EIF-4E, it was concluded that abundant EIF-4E in the testis may play an important role in spermatogenesis through translational regulation of stage-specific mRNAs during germ cell development.
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