טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentGoldberg Cohen Ilana
SubjectThe Role of the 3'UTR in the VEGF mRNA Turnover
DepartmentDepartment of Medicine
Supervisor Professor Andrew Peter Levy


Abstract

VEGF is a key mediator of hypoxia-induced angiogenesis and is regulated by hypoxia at the level of its mRNA.

We have identified a 40bp element in the 3'UTR of VEGF mRNA that increases VEGF mRNA stability under hypoxic conditions as well as with the binding of the protein HuR.  Characterization of HuR binding was facilitated by complementary in vivo and in vitro assays. Responsiveness to hypoxia was demonstrated with incubation of 293 cells under hypoxic or normoxic conditions.

RNase T1 protection and lead footprinting assays permitted identification of the precise HuR binding site. 

Deletion of this HuR binding site resulted in the transformation of the stability element to an instability element.

We identified a minimal instability element of 17bp that can confer significant mRNA destabilization in vivo when inserted in the 3’UTR of an RNA. UV cross-linking experiments identified the specific binding of 70Kd and 50Kd protein complexes to this 17bp element.  Destabilization conferred by the element in vivo and binding of the cytosolic protein complexes to the element were

markedly reduced by mutations within the 17bp sequence. Notably, mutation of an AUUUG sequence to AUUUA resulted in a total loss of destabilizing activity, thus, this 17 bp element is a novel instability element distinct from the AUUUA motif. We suggest a model whereby this 17bp element regulates the stability of VEGF mRNA by its recognition by ribonucleases that cause its rapid

degradation. However, under hypoxic conditions, HuR binding prevents its cleavage by RNases and increases VEGF mRNA stability.