טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentRachel Anunu
SubjectCharacterization of the Factors Affecting VEGF's IRES
Activity
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professor Levi Ben-Zion


Abstract

Vascular Endothelial Growth Factor (VEGF) has a central role in normal as well as in tumor angiogenesis. As such, VEGF is subjected to multi-level regulation at the transcriptional, post-transcriptional, translational and post-translational levels to ensure a proper expression during embryogenesis and edulthood. It’s mRNA contains an exceptionally long (1038bp) 5’ Untranslated Region (5’UTR), which has a role in transcriptional as well as translational regulation of VEGF. Recently we provided evidence showing that an Open Reading Frame (ORF) present in the 5’UTR, encodes for a new isoform of VEGF due to alternative translation initiation from CUG codon. The longest isoform that has addition of 180 amino acids in its 5’ end termed Long VEGF (L-VEGF). The 5’UTR contains two putative IRES elements which allow efficient translation even when cap dependent translation arrests.  We noticed that the 5’UTR  mediates efficient translation in mammalian cells and inhibits translation in virto. This study shows that there are RNA binding proteins that necessary for the 5’UTR activity in vitro. The proteins do not interact with IRES A. In addition we tested the interplay between IRES A and IRES B and show that IRES B is the dominant element in mediating translation from the authentic AUG but IRES A holds an important role as well. Using monocistronic vectors containing hairpin structure, we demonstrate that VEGF translation is mediated mainly by IRES mechanism. This research shows that  the uORF do not affect the expression from the  authentic AUG.