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