Biogenesis of Short Intronic Repeat 27-Nucleotide Small RNA from Endothelial Nitric-oxide Synthase Gene

Ming-Xiang Zhang,Cheng Zhang,Ying H Shen,Jian Wang,Xiao Nan Li,Yun Zhang,Joseph Coselli,Xing Li Wang

doi:10.1074/jbc.m801933200

Abstract

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.

Highlights

Ulation, arterial wall remodeling, and establishment of collateral circulation [1]
A recent Framingham Study reported no association between 33 single nucleotide polymorphisms in the Endothelial nitric-oxide synthase (eNOS) gene and endothelial function [7], others reported positive associations between the promoter T-786C polymorphism, CA repeats in intron 13, E298D at exon 7, and endothelial function and vascular diseases [8]
Association between the Expressions of the 27-nt sir-RNA and eNOS—Because 27nt sir-RNA is likely derived from the eNOS gene, we first evaluated the possible relationship in the expression between the two

Summary

Introduction

Ulation, arterial wall remodeling, and establishment of collateral circulation [1]. Effects of NO result either directly from reactions between NO and specific biological molecules through S-nitros(yl)ation [2], or indirectly from reactive nitrogen oxide species through oxidation [3]. Reduction in basal NO release may predispose to hypertension, thrombosis, vasospasm, and atherosclerosis [5], and overproduction of NO can result in excessive oxidative stress and inflammation, both of which promote vascular diseases. Central to all these physiological functions is the proper eNOS expression and adequate eNOS enzyme activity. Functional roles of the eNOS DNA variants are not clear, hundreds of studies investigating associations between various eNOS polymorphisms and vascular diseases have been published over the last decade. We speculate that the intron 4-derived 27-nt small RNA may function as a negative feedback regulator and maintain the stable transcription of eNOS

Methods

Results

Conclusion