After fertilization of sea urchin eggs, eIF4G is post-translationally modified and associated with the cap-binding protein eIF4E.
Release of eukaryotic initiation factor 4E (eIF4E) from its translational repressor eIF4E-binding protein (4E-BP) is a crucial event for the first mitotic division following fertilization of sea urchin eggs. Finding partners of eIF4E following fertilization is crucial to understand how eIF4E functions during this physiological process. The isolation and characterization of cDNA encoding Sphaerechinus granularis eIF4G (SgIF4G) are reported. mRNA of SgIF4G is present as a single 8.5-kb transcript in unfertilized eggs, suggesting that only one ortholog exists in echinoderms. The longest open reading frame predicts a sequence of 5235 nucleotides encoding a deduced polypeptide of 1745 amino acids with a predicted molecular mass of 192 kDa. Among highly conserved domains, SgIF4G protein possesses motifs that correspond to the poly(A) binding protein and eIF4E protein-binding sites. A specific polyclonal antibody was produced and used to characterize the SgIF4G protein in unfertilized and fertilized eggs by SDS-PAGE and western blotting. Multiple differentially migrating bands representing isoforms of sea urchin eIF4G are present in unfertilized eggs. Fertilization triggers modifications of the SgIF4G isoforms and rapid formation of the SgIF4G-eIF4E complex. Whereas rapamycin inhibits the formation of the SgIF4G-eIF4E complex, modification of these SgIF4G isoforms occurs independently from the rapamycin-sensitive pathway. Microinjection of a peptide corresponding to the eIF4E-binding site derived from the sequence of SgIF4G into unfertilized eggs affects the first mitotic division of sea urchin embryos. Association of SgIF4G with eIF4E is a crucial event for the onset of the first mitotic division following fertilization, suggesting that cap-dependent translation is highly regulated during this process. This hypothesis is strengthened by the evidence that microinjection of the cap analog m(7)GDP into unfertilized eggs inhibits the first mitotic division.
Oulhen
Nathalie
N
Université Pierre et Marie Curie-Paris6, UMR 7150, Equipe Cycle Cellulaire et Développement and CNRS, UMR 7150, Station Biologique de Roscoff, 29682 Roscoff CEDEX, France.
Salaün
Patrick
P
Cosson
Bertrand
B
Cormier
Patrick
P
Morales
Julia
J
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D039561:Eukaryotic Initiation Factor-4E / Q000302:isolation & purification / Q000378:metabolism*; D039603:Eukaryotic Initiation Factor-4G / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005306:Fertilization / Q000502:physiology*; D005982:Glutathione Transferase / Q000378:metabolism; D008969:Molecular Sequence Data; D010063:Ovum / Q000378:metabolism*; D010442:Peptide Chain Initiation, Translational; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012617:Sea Urchins / Q000166:cytology / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase