Internal Translation Initiation Mediated by the Angiogenic Factor Tie2

Eun-Hee Park,Joseph M Lee,Jaime D Blais,John C Bell,Jerry Pelletier

doi:10.1074/jbc.m412744200

Eun-Hee Park, Joseph M Lee + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m412744200

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Abstract

Tie2 is an endothelium-specific receptor tyrosine kinase required for normal blood vessel maturation. We report that Tie2 mRNA translation is maintained under hypoxic conditions. To identify the mechanism responsible for this, we undertook structure/function analysis of the Tie2 5'-untranslated region (UTR). Transcription start site mapping indicates the existence of a several mRNA isoforms containing unusually long 5'-UTRs (>350 nucleotides) with five upstream open reading frames. We find internal ribosome binding activity that allows the Tie2 mRNA to initiate in a cap-independent fashion. Our data provide a framework for understanding how Tie2 mRNA is translated despite a cumbersome structured 5'-UTR and how its production is secured under unfavorable environmental conditions.

Highlights

Angiogenesis is an essential step in allowing tumors to grow beyond 1–2 mm in diameter [1]
Tie2 mRNA Is Translated under Hypoxic Conditions—To assess the translational behavior of Tie2 mRNA under hypoxic conditions, we monitored its distribution across polysomes from normoxic and hypoxic treated primary human umbilical vein endothelial cells (HUVECs) (Fig. 1)
Quantitative Reverse Transcriptase (RT)-PCR from fractions collected from the bottom or top half of the polysomes (N/H-4 and N/H-3, respectively), as well as from the top of the gradient (N/H-1) and the region containing the ribosomal subunits (N/H-2), revealed that the distribution of Tie2 and VEGR mRNAs remain unchanged when cells are transferred from normoxic to hypoxic conditions (Fig. 1B)

Summary

EXPERIMENTAL PROCEDURES

Cell Lines, Transfections, and Hypoxia Treatment—Primary human umbilical vein endothelial cells (HUVECs) were purchased from Clonetics (Walkersville, MD) and maintained in endothelial growth medium-2 supplemented with growth factors, 2% fetal bovine serum, 50 ␮g/ml gentamicin, and 50 ␮g/ml amphotericin according to the manufacturer’s instructions

DNA transfections into primary HUVECs were performed using

RESULTS

DISCUSSION