Cap-Independent Translation Regulation of VEGF Using CRISPR-dCas13d
Vascular endothelial growth factor (VEGF) is a central driver of pathological angiogenesis in cancer and retinal disease. A G-quadruplex (GQ) structure that forms in an Internal Ribosome Entry Site (IRES) of VEGF was proposed to recruit the 40S ribosome and initiate cap-independent translation under hypoxia. We demonstrate that blocking this GQ with dCas13d represses VEGF translation by up to…
Vascular endothelial growth factor (VEGF) plays a crucial role in promoting angiogenesis in cancer and retinal diseases. A G-quadruplex (GQ) structure within VEGF's Internal Ribosome Entry Site (IRES) initiates cap-independent translation under hypoxic conditions. Researchers have discovered that blocking this GQ with a dCas13d protein suppresses VEGF translation by up to 5-fold, without affecting mRNA levels.
This phenomenon was observed in HeLa and human umbilical vein endothelial cells (HUVECs) through immunofluorescence, western blot assays, and bicistronic dual-luciferase experiments. Single molecule Forster resonance energy transfer (smFRET) and fluorescence enhancement assays confirmed the destabilization of the GQ by CRISPR-dCas13d.
The study also investigated the functional implications of this regulation through a tube formation assay in bovine aortic endothelial cells (BAOECs), yielding consistent results with VEGF expression levels. The findings demonstrate the potential of CRISPR-dCas13d as a precise and temporary translation regulator for genes containing translationally active secondary structures.
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