Delta-like 4 mRNA is regulated by adjacent natural antisense transcripts
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Recent evidence suggests that a majority of RNAs in the genome do not code for proteins. They are located in the sense (S) or antisense (AS) orientation and, to date, the functional significance of these non-coding RNAs (ncRNAs) is poorly understood. Here, we examined the relationship between S and AS transcripts in the regulation of a key angiogenesis gene, Delta-like 4 (Dll4).
Rapid Amplification of cDNA Ends (RACE) method was used to identify natural antisense transcripts in theDll4gene locus in murine and human endothelial cells, referred to as Dll4 Anti-Sense (Dll4-AS). Messenger RNA (mRNA) levels ofDll4andDll4-ASwere quantified by real-time PCR. The function ofDll4-ASwas investigated by overexpression and knocking down ofDll4-AS.
Dll4-AScomprises of three isoforms that map proximal to theDll4promoter region. Expression patterns ofDll4-ASisoforms vary among different endothelial cell lines, but are always congruent with those ofDll4. A dual promoter element in theDll4locus has been identified that controls the expression of both transcripts. BothDll4-ASandDll4are sensitive to cellular density in that higher cellular density favors their expression. ExogenousDll4stimuli such as VEGF, FGF and Notch signaling inhibitor altered bothDLL4-ASandDLL4expression suggesting co-regulation of the transcripts. Also, knocking down ofDll4-ASresults in down-regulation ofDll4expression. As a consequence, endothelial cell proliferation and migration increases in vitro, and sprout formation increases. The regulation ofDll4byDll4-ASwas also conserved in vivo.
A novel form of non-coding RNA-mediated regulation at the Dll4locus contributes to vascular developmental processes such as cell proliferation, migration and sprouting.