Abstract 1496: Biogenesis of Short Intronic Repeat 27nt Small RNA from Endothelial Nitric Oxide Synthase Gene
Previously, our studies have shown that the 27nt repeat polymorphism in endothelial nitric oxide synthase (eNOS) gene intron 4 is associated with an increased risk of vascular disease, eNOS expression and vascular nitric oxide (NO) production. We further discovered that the 27nt repeats in the eNOS intron 4 can be transcribed into 27nt small RNA, which can directly inhibit eNOS expression. Our earlier efforts have been tempted to classify this short intronic repeat derived small RNA (sir-RNA) as microRNA. However, more experimental evidence strongly suggests that the sir-RNA may either be a new class of small RNA or an atypical form of microRNA. In the present study, we characterized the biogenesis of the 27nt sir-RNA. Using Northern blot, we showed that the eNOS-derived 27nt sir-RNA expressed only in the eNOS expressing cells - endothelium and exclusively within nucleus. Cells containing 5×27nt repeats produced more 27nt sir-RNA and lower eNOS mRNA than the cells with 4×27nt repeats. Using pCMV-eNOS cDNA construct containing 5× and 10×27nt repeats, we demonstrated a dose-dependent effect in 27nt sir-RNA biogenesis. When the splicing junctions were mutated, biogenesis of the 27nt sir-RNA production was abolished. By cloning the DNA fragments upstream of the 27nt repeats, our experiments indicated that the 27nt sir-RNA biogenesis was unlikely driven by an independent promoter. By suppressing the expression of RNA polymerase type III Drosha or cytoplasmic type III RNAse Dicer - both of which are involved in small RNA processing, significantly reduced the biogenesis of the 27nt sir-RNA. In summary, our study suggests that the 27nt sir-RNA may have derived through eNOS pre-mRNA splicing and may represent a new class of small RNA. The more eNOS is transcribed, the more 27nt sir-RNA is produced, which specifically inhibits eNOS expression and functions as a negative feedback self-regulator. This may represent a novel molecular model in modifying the quantitative gene expression.
(Projected is supported by R01 HL66053; P50HL083704; AHA 0400031N)