Ribosomal scanning on the highly structured insulin-like growth factor II-leader 1

• Published in: The international journal of biochemistry & cell biology Vol. 34; no. 3; pp. 286 - 297
• Main Authors: van der Velden, Alike W., van Nierop, Kirsten, Voorma, Harry O., Thomas, Adri A.M.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 01.03.2002
• Subjects: 5' Untranslated Regions - genetics; Animals; COS Cells; Genes, Reporter; Growth factor; Hairpin; Humans; Insulin-Like Growth Factor II - genetics; Insulin-Like Growth Factor II - metabolism; Nucleic Acid Conformation; Peptide Chain Initiation, Translational; Ribosomes - metabolism; Scanning; Translation inı̈tiation; eIF, eukaryotic initiation factor; GFP, green fluorescent protein; Scanning; uAUG, upstream AUG; ORF, open reading frame; EMCV, encephalomyocarditis virus; sHP, short hairpin; HP, hairpin; Hairpin; wt, wild type; nt, nucleotide(s); 5′UTR, 5′ untranslated region; IGF, insulin-like growth factor; sAUG, start AUG codon; Translation inı̈tiation; CAT, chloramphenicol acetyl transferase; IGFl1, insulin-like growth factor II-leader 1; IRES, internal ribosomal entry site; Growth factor
• ISSN: 1357-2725; 1878-5875
• DOI: 10.1016/S1357-2725(01)00116-9

The complex architecture of human insulin-like growth factor (IGF) II-leader 1 of 592 nucleotides (nt), with one open reading frame (ORF), and the potential to fold into stable structures makes efficient linear ribosomal scanning difficult to comprehend. Indeed, leader 1-driven reporter expression is low in rabbit reticulocyte lysate. Contrarily, leader 1 is very efficient in cells. Therefore, we tested whether this 5′UTR uses an alternative mechanism for translation initiation in vivo, internal entry or ribosomal shunting. Internal initiation was tested by introducing leader 1 into the intercistronic region of a bicistronic vector. Second cistron expression, driven by leader 1, was lower than by the intercistronic β-globin 5′UTR, indicating that leader 1 does not contain an internal ribosomal entry site (IRES). Shunting was tested by inserting hairpin (HP) structures, capable of blocking ribosomal scanning, at eight positions in leader 1. After transfection, these mutant 5′UTRs were incapable of directing reporter expression. Less stable HPs at the same positions increased the activity to 50% of wild-type activity, indicating that insertions at these positions are not disastrous for initiation. These data indicate that the translational machinery encounters major parts of leader 1. As scanning seems unlikely, and internal entry and shunting were shown not to occur, we discuss a modified scanning mechanism for architecturally complex 5′UTRs.