Delta-like 4 mRNA is regulated by adjacent natural antisense transcripts

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• The majority of the human genome transcribes non-coding RNAs (ncRNAs), but their functions remain largely unknown.
• ncRNAs can exist in sense (S) or antisense (AS) orientations relative to protein-coding genes.
• The role of ncRNAs in regulating key developmental genes, such as those involved in angiogenesis, is an area of active research.

Purpose of the Study:

• To investigate the relationship between sense and antisense transcripts in the regulation of the Delta-like 4 (Dll4) gene.
• To identify and characterize natural antisense transcripts at the Dll4 locus.
• To elucidate the functional significance of Dll4 antisense transcripts (Dll4-AS) in endothelial cell biology and vascular development.

Main Methods:

• Rapid Amplification of cDNA Ends (RACE) was employed to identify Dll4-AS transcripts in murine and human endothelial cells.
• Quantitative real-time PCR was used to measure messenger RNA (mRNA) levels of Dll4 and Dll4-AS.
• Functional studies involved overexpression and knockdown of Dll4-AS to assess its impact on Dll4 expression and cellular functions.

Main Results:

• Three isoforms of Dll4-AS were identified, mapping near the Dll4 promoter and exhibiting congruent expression patterns with Dll4.
• A dual promoter element controls the co-expression of Dll4 and Dll4-AS, both influenced by cellular density and signaling pathways (VEGF, FGF, Notch).
• Knockdown of Dll4-AS led to decreased Dll4 expression, resulting in increased endothelial cell proliferation, migration, and in vitro/in vivo sprout formation.

Conclusions:

• A novel mechanism of non-coding RNA-mediated regulation at the Dll4 locus has been discovered.
• Dll4-AS plays a crucial role in controlling Dll4 expression, thereby influencing vascular development.
• This ncRNA-mediated regulation contributes to fundamental vascular processes including cell proliferation, migration, and sprouting.