Regulation of endothelial nitric oxide synthase by small RNA

Abstract

Repeats (27-nt) in intron 4 have been shown to play a cis-acting role in endothelial nitric oxide synthase (eNOS) promoter activity. We hypothesize that the 27-nt repeats could be the source of small nuclear RNA specifically regulating eNOS expression. In this study, we used synthesized 27-nt RNA duplex and found that the eNOS gene transcriptional efficiency was reduced 63% (0.047 ؎ 0.009 vs. 0.126 ؎ 0.015, P < 0.01) by nuclear run-on assay. In endothelial cells transfected with the 27-nt small RNA duplex, we found that the eNOS mRNA and protein levels were decreased by >64% (P < 0.01). Conversely, a randomly selected 27-nt from luciferase gene had no effect on the eNOS expression. Furthermore, this eNOS silencing effect appeared to be reversible under the stimulation of vascular endothelial growth factor (10 ng͞ml), which is known to upregulate eNOS expression. Using in situ hybridization and Northern blotting, we observed the presence of endogenous eNOS intron 4-derived 27-nt small RNA, which was confined to the nucleus. In summary, we demonstrated that intron-based microRNAs in eNOS can induce significant gene specific transcriptional suppression, which could be an effective negative feedback regulator for gene expression.intron ͉ microRNA ͉ negative feedback regulation ͉ repeat polymorphism M ost eukaryotic genes are interrupted by one or more introns, which must be removed in the process of pre-mRNA splicing within the nucleus. The mature mRNA with intact ORF is then exported to the cytoplasm for translation. Since the discovery of introns as the noncoding sequences (1), they have mostly been regarded as redundant and useless genomic sequences. However, with the advancement in molecular technology, these intervening sequences have recently been recognized to play a significant role in gene evolution and expression (2-4). Introns can function as enhancer͞repressor to regulate cell specific gene transcription; they can modulate the spliceosome conformation and affect the efficiency of pre-mRNA splicing (5, 6). A recent study has shown that the expression of human ␤-globin gene is highly intron-dependent, which regulates both pre-mRNA splicing and translational efficiency (7). Moreover, the intronic binding site for nuclear proteins mediates the p53-dependent transcriptional activation (8); intronic regulatory elements confer to control developmental and cellspecific gene expression (9). Thus, it is clear that introns are not only essential for genomic integrity, but are also important for controlling cell-specific gene expression.Of particular interest, Hui et al. (10) have recently shown that the variable-length CA repeats in the endothelial nitric oxide synthase (eNOS) intron 13 can bind with hnRNP and affect eNOS splicing. Studies of ours and others have further suggested that the 27-nt repeats in the eNOS intron 4 could be potentially functional in regulating eNOS expression (11,12). We have shown that the 27-nt repeats in intron 4 could act as a possible enhancer͞repressor for the eNOS transcription (13...

The BCL2 major breakpoint region (mbr) regulates gene expression

“…To distinguish these two possibilities, the transcriptional activities of wildtype and targeted BCL2 alleles were examined with nuclear run-on transcription analysis using a modified protocol with biotin-16-uridine triphosphate (UTP) (Patrone et al, 2000;Tong et al, 2005;Zhang et al, 2005). Results of nuclear run-on assays showed that the transcriptional activity of the targeted allele was 10.3-fold lower than that of the wild-type allele, indicating the expression discrepancy of the wild-type and the targeted BCL2 alleles was mainly due to the difference in transcriptional activities between the two alleles (DCT ¼ 34.13-30.77E3.36; 2 3.36 ¼ 10.3) (Figure 4).…”

“…Nuclear run-on assay The transcriptional activity of wild type and of targeted BCL2 alleles was determined by nuclear run-on transcription analysis according to a modified protocol that uses biotin-16-uridine triphosphate (UTP) (Roche, Sandhofer Strasse, Mannheim, Germany) (Patrone et al, 2000;Tong et al, 2005;Zhang et al, 2005). Briefly, 2-3 Â 10 7 Nalm6-1 cells were washed twice with ice-cold PBS and resuspended in cell lysis buffer containing 10 mM Tris-HCl (pH 7.4), 3 mM MgCl 2 , 10 mM NaCl, 150 mM sucrose and 0.5% NP-40.…”

MicroRNA-directed transcriptional gene silencing in mammalian cells

“…2B). Nuclear run-on experiments (18) in HEK-293 cells, performed at the 20-and 40-h time points after transfection of control or miR-320 duplexes, indicated that increased levels of mature miR-320 induced TGS of the POLR3D gene (Fig. 2C).…”

Influence of Endothelial Nitric Oxide Synthase Gene Intron-4 27bp Repeat Polymorphism on Its Expression in Autoimmune Diseases

“…Recently, Zhang et al have shown that the 27 bp repeats in eNOS intron 4 produce small 27 bp micro RNAs [37,38]. This intron-based micro RNA can induce gene specific transcriptional suppression by modifying histone acetylation and DNA methylation, which may serve as an effective negative feedback regulator of eNOS expression.…”