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 m7GTP cap structure of eukaryotic mRNAs and influences the overall rates of translation. The eIF4E protein is subject to regulation at a number of levels that allow it to modulate translation of maternal mRNAs in early embryos before the onset of zygotic transcription. In zebrafish eIF4E (zeIF4E) mRNA levels are elevated in specific tissues and at specific times during embryogenesis. We have characterized the organization of the zeIF4E gene to facilitate elucidation of the molecular mechanisms that influence its expression. The zeIF4E gene spans about 14 kb and like its human counterpart is comprised of seven exons. Alternative splicing between the first and second exon generates two mRNA splice-forms called SF1 and SF2. Nuclease-S1-protection and primer-extension reveal two zeIF4E transcriptional start-sites. Transcripts initiating from the distal start-site during oogenesis are exclusively SF1, while initiation 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 inhibits in vitro protein synthesis in a concentration-dependent manner, suggesting it functions as a translational attenuator. Thus, specific transcriptional activation from the distal start-site may provide a unique mechanism for transcriptional regulation of the levels, as well as the function of zeIF4E mRNAs.
Fahrenkrug
S C
SC
Department of Genetics, Cell Biology and Development, University of Minnesota, St. Paul 55108-1095, USA.
Joshi
B
B
Hackett
P B
PB
Jagus
R
R
D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D039561:Eukaryotic Initiation Factor-4E; D005091:Exons / Q000235:genetics; D015698:Genomic Library; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D023282:Nuclease Protection Assays; D010448:Peptide Initiation Factors / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D012315:RNA Caps / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*; D015027:Zebrafish / Q000235:genetics* / Q000378:metabolism
D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors; D012315:RNA Caps; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D015719:Single-Strand Specific DNA and RNA Endonucleases