טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentEyal Scheinman
SubjectFactors Affecting the Transcription of VEGF
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professor Levi Ben-Zion


Abstract

VEGF is a potent angiogenic agent, which acts as a specific mitogen for vascular endothelial cells. At least 5 isoforms of VEGF are generated due to alternative splicing. Recently we have shown that the very long 5’UTR (1038bp) of VEGF has an open reading frame which is initiated from alternative translation start site that adds 180 aa infront of the initiation AUG. My first goal was to identify alternative upstream start codons and sites which are important to mRNA secondary structure stability. Using point mutation along the 5’UTR mRNA and computer analysis, we could determine that the area responsible for the 5’UTR mRNA structure stability starts at CUG1 and ends at GUG3 not including CUG3. The area starting at ACG2 and ending at the AUG site found to be less sensitive to point mutations regarding the affect on the structure stability of the 5’UTR mRNA.

VEGF also possesses a very long 3’UTR containing AU rich elements. The 3’UTR of VEGF is responsible for the stability and instability of the mRNA. My second goal was to determine the effect of the 3’UTR on the translation pattern under different physiological conditions. To achieve the this goal we cloned the 3’UTR of VEGF and tested its effect together with HuR on translation of VEGF. From our findings we concluded that elongated isoforms as well as the main protein are translated under normal conditions, while under hypoxic conditions the 3’UTR directs the 43S ribosomal subunit toward the AUG start codon to form the main protein. Therefore it is suggested that elongated isoforms have either role in regulation of VEGF under normal conditions since they where found in the nucleus, or they function as storage molecules of inactive VEGF.