A-to-I RNA Editing Regulates Gene Expression and Controls Angiogenesis in Human Endothelial Cells

Background: The enzymes ADAR1 and ADAR2 (adenosine deaminase acting on RNA-1 and -2) catalyze adenosine-to-inosine (A-to-I) RNA editing, which is an important posttranscriptional regulation mechanism. The objectives of the present study are to identify the extent of ADAR1/ADAR2-mediated RNA editing of mRNAs in endothelial cells and its implications in angiogenesis.

Methods and Results: The members of the ADARs family ADAR1 and ADAR2 are highly expressed on mRNA and protein level in human venous umbilical endothelial cells (HUVECs). With the help of high-throughput RNA-sequencing and bioinformatics analyses, we indentified more than 5,000 A-to-I editing events in Alu regions of mRNAs in HUVECs treated with scrambled oligonucleotides. ADAR1 knockdown resulted in a profound reduction of RNA editing events, whereas siADAR2 had no effects. Transcriptome expression analysis revealed that ADAR1 knockdown critically affected the expression of 134 mRNAs. Among the most extensively edited and regulated mRNAs with a known important function in vascular system was cathepsin S (CTSS). We observed a cluster of RNA editing sites at the 3’ UTR of CTSS mRNA, which were validated by cloning/Sanger sequencing. ADAR1 knockdown abolished the editing of the 3’UTR and resulted in a 50%-downregulation of mRNA and protein expression of CTSS. Next, we tested in silico the effect of RNA editing on RNA secondary structure by entering 57-nt sequence of the 3’ UTR of CTSS into two different RNA folding programs (m-fold and RNA fold web server). We observed that A-to-I RNA editing of the 3’ UTR of CTSS is predicted to result in a profound change of the RNA secondary structure. Since CTSS is critically involved in angiogenesis, we studied the effect of ADAR1 knockdown in endothelial cell sprouting. ADAR1 knockdown by three different siRNAs results in significant inhibition of angiogenic sprouting of endothelial cells (52.3±8.2 % inhibition).

Conclusions: ADAR1 is the main RNA editing enzyme in human endothelial cells. ADAR1 regulates the expression of CTSS and controls angiogenesis. Ongoing studies address the effect of ADAR1-mediated RNA editing in posttranscriptional regulation of CTSS and its role in neovascularization in vivo.