The Tie2 5' untranslated region is inhibitory to 5' end-mediated translation initiation

Tie2 is an endothelium-specific receptor tyrosine kinase required for normal blood vessel maturation, remodeling, and stability. Tie2 expression is also upregulated in various cancers implicating a role in tumor angiogenesis. Its mRNA transcript contains an unusually long (372 nucleotides) 5' untranslated region (UTR) with five upstream open reading frames (uORFs) and an internal ribosome entry site (IRES) that allows this mRNA to be translated under hypoxic conditions. This sets up an alternative initiation pathway with the potential to clash with 5' end-mediated initiation from the same template. Herein, we define experimental conditions under which the Tie2 IRES is not active, allowing us to assess the contribution of the 5' UTR to cap-dependent translation on the Tie2 transcript. We find that the Tie2 5' UTR is inhibitory to translation initiation with ribosome flow decreasing following encounters with each uORF. No single uORF was found to harbor significant cis-acting inhibitory activity. Our results suggest that the uORFs within the Tie2 5' UTR serve to decrease the percent of ribosomes competent for reinitiation as these traverse the mRNA 5' UTR, thus minimizing interference with the IRES.