PROTEINS BINDING TO THE LEADER OF THE 6.0-KB MESSENGER-RNA OF HUMAN INSULIN-LIKE GROWTH-FACTOR-2 INFLUENCE TRANSLATION

The leader of the 6.0 kb human insulin-like growth factor 2 (IGF-2) mR NA, leader 3, has been reported to partially repress translation. In t he regulation of this phenomenon, RNA-binding proteins may play a role . Using UV-irradiation crosslinking, we found specific binding of four proteins (57, 43, 37 and 36 kDa) to this leader. Binding of these pro teins to RNA proved to be highly sensitive to the potassium chloride c oncentration in the buffer solution, each protein having its own optim um. The 57 kDa protein was indistinguishable by size, binding properti es and immunoprecipitation from the polypyrimidine tract binding prote in (PTB), first described as a nuclear protein binding to the polypyri midine tracts (PPTs) in introns. Cross-competition experiments showed that leader 3 has a much higher affinity for this 57 kDa protein than the PPT on which PTB was originally characterized. By competition with different fragments of leader 3, we were able to localize the binding of the 57 kDa protein to a 162 nt RNA fragment (AsnI-PvuII) in the 3' -part of the leader. When placed before a chloramphenicol acetyltransf erase (CAT) open reading frame, this RNA fragment stimulated translati on in reticulocyte lysate 3-fold, while other fragments of leader 3 re pressed translation. The efficient translation directed by the 162 nt AsnI-PvuII fragment fused to CAT could be repressed by adding free Asn I-PvuII RNA fragment, indicating that the high translation efficiency of the AsnI-PvuII-CAT synthetic mRNA was due to the binding of protein and not to the structure of the RNA itself.