Two haplotypes of 5' untranslated region in L-myc gene with different internal ribosome entry site activity

We found two polymorphisms, A or C, at position 63 from the L-myc transcription start site, and 8- or 9-base Gs stretch between positions 82 and 89 or 90, in L-myc 5' UTR, which exhibited a strong linkage disequilibrium as two haplotypes, A-9G and C-8G. We analyzed whether both haplotypes of 5' UTR of the L-myc gene, each of which is approximately 200 nt long, are involved in translational regulation of the L-myc gene. Each haplotype of 5' UTR was inserted between Renilla luciferase and firefly luciferase genes to construct a bicistronic vector which was transcribed as a single mRNA. A translation of the downstream cistron was increased 70-100-fold in cos 7 cells, and 7-10-fold in HeLa S3 cells compared with a control vector without insertion. However, in this bicistronic assay system, both haplotypes of the L-myc 5' UTR showed similar internal ribosome entry site (IRES) activity. When a stable hairpin loop with a palindromic sequence was inserted into a 5'-end of bicistronic mRNA, the hairpin loop decreased only the activity of an upstream luciferase in both cos 7 and HeLa S3 cells, supporting the presence of an IRES. We also inserted 5' UTR of the L-myc gene into the 5'-end of reporter mRNA. C-8G showed approximately 2-fold higher IRES activity than A-9G in both cos 7 and HeLa S3 cells, suggesting that two polymorphisms in the L-myc exon 1 might be involved in regulation of L-myc protein expression.