Alternative transcriptional initiation and splicing define the translational efficiencies of zebrafish mRNAs encoding eukaryotic initiation factor 4E

Translation initiation factor 4E (eIF4E) binds to the m(7)GTP cap structure of eukaryotic mRNAs and influences the overall rates of translation. The e IF4E protein is subject to regulation at a number of levels that allow it t o modulate translation of maternal mRNAs in early embryos before the onset of zygotic transcription. In zebrafish eIF4E (zeIF4E) mRNA levels are eleva ted in specific tissues and at specific times during embryogenesis. We have characterized the organization of the zeIF4E gene to facilitate elucidatio n of the molecular mechanisms that influence its expression. The zeIF4E gen e spans about 14 kb and like its human counterpart is comprised of seven ex ons. Alternative splicing between the first and second exon generates two m RNA splice-forms called SF1 and SF2. Nuclease-S1-protection and primer-exte nsion reveal two zeIF4E transcriptional start-sites. Transcripts initiating from the distal start-site during oogenesis are exclusively SF1, while ini tiation from the proximal start-site generates both splice-forms. Although translation in vitro of SF1 mRNA gives rise to a protein consistent in mass with affinity-purified zeIF4E, SF2 mRNA does not. Instead, SF2 mRNA inhibi ts in vitro protein synthesis in a concentration-dependent manner, suggesti ng it functions as a translational attenuator. Thus, specific transcription al activation from the distal start-site may provide a unique mechanism for transcriptional regulation of the levels, as well as the function of zeIF4 E mRNAs.