Proteins binding to the leader of the 6.0 kb mRNA of human insulin-like growth factor 2 influence translation.
The leader of the 6.0 kb human insulin-like growth factor 2 (IGF-2) mRNA, leader 3, has been reported to partially repress translation. In the 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 proteins to RNA proved to be highly sensitive to the potassium chloride concentration in the buffer solution, each protein having its own optimum. The 57 kDa protein was indistinguishable by size, binding properties and immunoprecipitation from the polypyrimidine tract binding protein (PTB), first described as a nuclear protein binding to the polypyrimidine 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 acetyltransferase (CAT) open reading frame, this RNA fragment stimulated translation in reticulocyte lysate 3-fold, while other fragments of leader 3 repressed translation. The efficient translation directed by the 162 nt AsnI-PvuII fragment fused to CAT could be repressed by adding free AsnI-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.
de Moor
C H
CH
Department of Pediatrics, Utrecht University, The Netherlands.
Jansen
M
M
Bonte
E J
EJ
Thomas
A A
AA
Sussenbach
J S
JS
Van Den Brande
J L
JL
D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D002474:Cell-Free System; D005091:Exons; D005796:Genes; D017930:Genes, Reporter; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000096:biosynthesis / Q000235:genetics*; D008113:Liver Neoplasms / Q000473:pathology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000187:drug effects*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D016601:RNA-Binding Proteins / Q000378:metabolism / Q000494:pharmacology*; D012156:Reticulocytes / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D016601:RNA-Binding Proteins; D007335:Insulin-Like Growth Factor II