Biogenesis of short intronic repeat 27-nucleotide small RNA from endothelial nitric-oxide synthase gene.
Endothelial nitric-oxide synthase (eNOS) is a constitutively expressed gene in endothelium that produces NO and is critical for vascular integrity. Previously, we reported that the 27-nucleotide (nt) repeat polymorphism in eNOS intron 4, a source of 27-nt small RNA, which inhibits eNOS expression, were associated with cardiovascular risk and expression of the eNOS gene. In the current study, we investigated the biogenesis of the intron 4-derived 27-nt small RNA. Using Northern blot, we showed that the eNOS-derived 27-nt short intronic repeat RNA (sir-RNA) expressed only in the eNOS expressing endothelial cells. Cells containing 10 x 27- or 5 x 27-nt repeats produced higher levels of 27nt sir-RNA and lower levels of eNOS mRNA than the cells with 4 x 27-nt repeats. The 27nt sir-RNA was mostly present within the endothelial nuclei. When the splicing junctions of the 27-nt repeat containing intron 4 in the full-length eNOS cDNA vector were mutated, 27nt sir-RNA biogenesis was abolished. Suppression of Drosha or Dicer diminished the biogenesis of the 27nt sir-RNA. Our study suggests that the 27nt sir-RNA derived through eNOS pre-mRNA splicing may represent a new class of small RNA. The more eNOS is transcribed or higher number of the 27-nt repeats, the more 27nt sir-RNA is produced, which functions as a negative feedback self-regulator by specifically inhibiting the host gene eNOS expression. This novel molecular model may be responsible for quantitative differences between individuals carrying different numbers of the polymorphic repeats hence the cardiovascular risk.
Zhang
Ming-Xiang
MX
Adult Section of Cardiothoracic Surgery, Texas Heart Institute at St Luke's Episcopal Hospital, Division of Cardiothoracic Surgery, Michael E DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX 77030, USA. mzhang1@bcm.edu
Zhang
Cheng
C
Shen
Ying H
YH
Wang
Jian
J
Li
Xiao Nan
XN
Zhang
Yun
Y
Coselli
Joseph
J
Wang
Xing Li
XL
D017398:Alternative Splicing / Q000235:genetics; D001483:Base Sequence; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007438:Introns / Q000235:genetics*; D008969:Molecular Sequence Data; D052250:Nitric Oxide Synthase Type III / Q000235:genetics* / Q000378:metabolism*; D009711:Nucleotides / Q000096:biosynthesis* / Q000235:genetics*; D009928:Organ Specificity; D011401:Promoter Regions, Genetic / Q000235:genetics; D012313:RNA / Q000096:biosynthesis* / Q000235:genetics*
D018076:DNA, Complementary; D009711:Nucleotides; D012313:RNA; D052250:Nitric Oxide Synthase Type III