Expression Of Sense RNA Research Articles

Alu antisense RNA ameliorates methylglyoxal-induced human lens epithelial cell apoptosis by enhancing antioxidant defense.

To determine whether an antisense RNA corresponding to the human Alu transposable element (Aluas RNA) can protect human lens epithelial cells (HLECs) from methylglyoxal-induced apoptosis. Cell counting kit-8 (CCK-8) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were used to assess HLEC viability. HLEC viability/death was detected using a Calcein-AM/PI double staining kit; the annexin V-FITC method was used to detect HLEC apoptosis. The cytosolic reactive oxygen species (ROS) levels in HLECs were determined using a reactive species assay kit. The levels of malondialdehyde (MDA) and the antioxidant activities of total-superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) were assessed in HLECs using their respective kits. RT-qPCR and Western blotting were used to measure mRNA and protein expression levels of the genes. Aluas RNA rescued methylglyoxal-induced apoptosis in HLECs and ameliorated both the methylglyoxal-induced decrease in Bcl-2 mRNA and the methylglyoxal-induced increase in Bax mRNA. In addition, Aluas RNA inhibited the methylglyoxal-induced increase in Alu sense RNA expression. Aluas RNA inhibited the production of ROS induced by methylglyoxal, restored T-SOD and GSH-Px activity, and moderated the increase in MDA content after treatment with methylglyoxal. Aluas RNA significantly restored the methylglyoxal-induced down-regulation of Nrf2 gene and antioxidant defense genes, including glutathione peroxidase, heme oxygenase 1, γ-glutamylcysteine synthetase and quinone oxidoreductase 1. Aluas RNA ameliorated methylglyoxal-induced increases of the mRNA and protein expression of Keap1 that is the negative regulator of Nrf2. Aluas RNA reduces apoptosis induced by methylglyoxal by enhancing antioxidant defense.

Anticubilin Antisense RNA Ameliorates Adriamycin-Induced Tubulointerstitial Injury in Experimental Rats

This study was designed to determine the effects of in vivo anticubilin antisense RNA on the uptake of albumin in tubules and on the tubulointerstitial injury in adriamycin-induced proteinuric rats. Adriamycin-treated rats were subjected to intrarenal delivery of adenoviral vectors encoding empty plasmid, cubilin sense RNA expression vector pAd-CUB or anticubilin antisense RNA expression vector pAd-ACUB on day 3. On days 14 and 28, half of the rats in each group were randomly selected to be killed, and blood samples, kidney tissues and 24-hour urine were collected. The diseased rats treated with pAdEasy-ACUB showed a 60% decrease in serum creatinine and glomerular filtration rate. Interestingly, the anticubilin antisense treatment led to a marked increase in albuminuria. Antisense treatment attenuated the histologic changes on both day 14 and day 28. The antisense treatment induced more than 60% recovery of adriamycin-induced injury, accompanied with 85% knockdown in the expression of cubilin protein and markedly decreased albumin deposition. Adriamycin induced an increase in the expression of monocyte chemoattractant protein-1, transforming growth factor-β and regulated on activation in normal T-cell expressed and secreted and the number of infiltrating cells, which was reversed by the antisense treatment. Anticubilin antisense RNA delivered by an adenoviral vector ameliorates albuminuria-induced glomerulosclerosis and tubulointerstitial damage in adriamycin nephrotic rats, indicating that cubilin could be a potential therapeutic target in proteinuric nephropathy.

Predominant Expression of Two Bovid-Specific Y-Chromosome Genes (ZNF280BY and PRAMEY) in Spermatids.

Autosome-to-Y transposition of male fertility genes is a recurrent theme in mammalian Y-chromosome evolution. As the transposition events occurred separately in different lineages, Y-chromosome genes have been accumulated in the male-specific region (MSY) in a lineage-dependent manner. We have recently discovered two bovid-specific Y-chromosome gene families, Zinc finger protein 280B Y-link (ZNF280BY) and preferentially expressed antigen in melanoma Y-linked (PRAMEY), derived from the transposition of a gene block on the bovine chromosome (BTA) 17. After the transposition, these two gene families were amplified differentially. Over 130 active ZNF280BY loci and 240 ZNF280BY pseudogenes are distributed over the entire MSY region, whereas 16 copies (8 active and 8 pseudogenes) of PRAMEY are present in a small region of MSY on the bovine Y chromosome. Although the function of the ZNF280B/ZNF280BY family is unknown in mammals, the Drosophila ortholog, suppressor of Hairy-wing [Su(Hw)], is a transcriptional regulator and is required for a functional retroviral gypsy insulator. The PRAME/PRAMEY family belongs to the cancer-testis antigens (CTAs) that are predominantly expressed in normal testis and a variety of tumors. PRAME functions as a transcriptional repressor in the retinoic acid receptor (RAR) signaling cascade, and the over-expression of PRAME results in tumorigenesis. Therefore, we hypothesized that ZNF280BY and PRAMEY may play crucial roles in bovine spermatogenesis and male fertility. As the first step to characterize the functions of these two gene families, we analyzed the expression patterns of both families by using RT-PCR, quantitative (q) RT-PCR, cRNA testis section in situ hybridization (ISH) and RNA-deep sequencing approaches. As expected, the expressions of these two Y-chromosome genes are predominant in bovine testes. The RT-PCR analyses revealed that ZNF280BY was expressed predominantly in the testis and slightly in liver, adrenal gland and lymph node, whereas the PRAMEY was expressed specifically in the testis. Furthermore, the sense and antisense RNAs of ZNF280BY and PRAMEY displayed distinct expression patterns both temporally and spatially. ISH with ZNF280BY probes indicated that the ZNF280BY sense RNA was widely expressed, but the antisense RNA was detected only in the spermatids. In contrast, the sense RNA of PRAMEY was expressed specifically in spermatids, whereas the antisense RNA was expressed in all cell types of the seminiferous tubules, with the highest expression in spermatids. qRT-PCR analysis indicated that the expression of ZNF280BY sense RNA increased significantly with age, while the ZNF280BY antisense RNA expression was stable during testis development. For PRAMEY, the expression of the sense RNA was low in 5 to 11-day and 3-month-old testes, but up-regulated in 8-month- and 24-month-old testes; the expression of antisense PRAMEY RNA increased slightly with age. Deep sequencing of the selected cDNAs further suggested that different loci of ZNF280BY and PRAMEY were expressed differentially. Conclusively, our results suggest that the transposed ZNF280BY and PRAMEY families have acquired functional and evolutionary advantages for male spermatogenesis during evolution in cattle. This project was supported by National Research Initiative Competitive Grants no. 2005-35205-18653 and no. 2010-65205-20362 from the USDA National Institute of Food and Agriculture to WSL. (platform)

Estrogen Down-regulation of the Scx Gene Is Mediated by the Opposing Strand-overlapping Gene Bop1

Recent genome-wide transcriptome studies suggest the presence of numerous bidirectional overlapping coding gene pairs in mammalian genomes. Various antisense RNAs are reported as non-coding RNAs that regulate the expression of sense RNA. However, it is still unclear whether the expression of bidirectional overlapping coding genes are regulated by the opposite strand gene transcript acting as a non-coding RNA. Bop1 and Scx are a pair of bidirectional overlapping coding genes related to cellular proliferation and differentiation, respectively. Scx gene is localized in the intron 3 region of the Bop1 gene. The expression of these genes is reciprocally regulated by estrogen (E2) in the mouse uterus. In situ hybridization indicated that both genes are expressed in the uterine endometrial epithelial cells and that the antisense RNA of Scx (Bop1 intronic RNA) accumulates as a stable RNA in these cells. The existence of Bop1 intronic RNA was confirmed by reverse transcription-PCR and was increased after E2 treatment, coinciding with a decrease in Scx mRNA. Murine myoblasts expressing doxycycline-inducible endogenous Bop1 gene showed an increase in Bop1 intronic RNA and a simultaneous decrease in Scx mRNA. Murine fibroblasts expressing Scx mRNA from an exogenous Scx mini-gene indicated that the accumulation of Bop1 intronic RNA impairs the Scx gene expression in a trans-acting manner, which resulted in the reduction of the Scx mRNA level. This study demonstrates a novel example of hormone-stimulated intronic non-coding RNA down-regulating the expression of an opposing strand-overlapping coding gene.

Differential Regulation of the Myosin Heavy Chain Genes α and β in Rat Atria and Ventricles: Role of Antisense RNA

The myosin heavy chain (MHC) genes are regulated by triiodothyronine (T3) in a reciprocal and chamber-specific manner. To further our understanding of the potential mechanisms involved, we determined the T3 responsiveness of the MHC genes, alpha and beta, and the beta-MHC antisense (AS) gene in the rat ventricles and atria. Hypothyroid rats were administered a single physiologic (1 microg) or pharmacologic (20 microg) dose of T3, and sequential measurements of beta-MHC hn- and AS RNA and alpha-MHC heterogeneous nuclear RNA from rat ventricular and atrial myocardium were performed with reverse transcription PCR. We have demonstrated that T3 treatment increases the myocyte content of an AS beta-MHC RNA in atria and ventricles that includes sequences complementary to both the first 5' and last 3' introns of the beta-MHC sense transcript. In the hypothyroid rat ventricle, beta-MHC sense RNA expression is maximal, while in the euthyroid rat ventricle, beta-MHC AS RNA is maximal. beta-MHC AS expression increased by 52 +/- 9.8% at the peak, 24 hours after injection of a physiologic dose of T3 (1 microg/animal), while beta-MHC sense RNA decreased by 41 +/- 2.2% at 36 hours, the nadir. In hypothyroid atria, beta-MHC AS RNA was induced by threefold within 6 hours of administration of 1 microg T3, demonstrating that in the atria, beta-MHC AS expression is regulated by T3, while alpha-MHC expression is not. In the hypothyroid rat heart ventricle, beta-MHC AS RNA expression increases in response to T3 similar to that of alpha-MHC. Simultaneous measures of beta-MHC sense RNA are decreased, suggesting a possible mechanism for AS to regulate sense expression. In atria, while alpha-MHC is not influenced by thyroid state, beta-MHC sense and AS RNA were simultaneously and inversely altered in response to T3. This confirms a close positive relationship between T3 and beta-MHC AS RNA in both the atria and ventricles, while demonstrating for the first time that alpha- and beta-MHC expression is not coupled in the atria.

Polypyrimidine tract-binding protein (PTB) inhibits Hepatitis C virus internal ribosome entry site (HCV IRES)-mediated translation, but does not affect HCV replication

Polypyrimidine tract-binding protein (PTB) has previously been shown to affect Hepatitis C virus (HCV) IRES-mediated translation. In the present study we investigated the functional role of PTB for HCV translation, replication and chronic HCV infection. Bicistronic HCV IRES reporter plasmids and two different subgenomic replicons (bicistronic: pHCVrep1bBB7 (s1179I); monocistronic: pFK1-389/hyg-ubi/NS3-3'/5.1) were used to analyze the effects of PTB. Following transfection of plasmids expressing PTB RNA in sense or antisense orientation, translational activity and HCV RNA were analyzed by luciferase assay, quantitative real-time RT-PCR and northern blot analysis. Additionally, in liver tissue (n = 53) intrahepatic PTB RNA levels were determined by quantitative real-time RT-PCR. Significant inhibition of HCV IRES activity up to 42.6% was observed upon PTB sense RNA expression for HCV IRES reporter plasmids, while translational activity was enhanced up to 63.8% for PTB antisense RNA expression. In the HCV replicons PTB did not affect replication and no correlation was found between intrahepatic PTB mRNA levels and serum HCV RNA or histological changes in liver tissue of HCV infected patients. Although PTB inhibits HCV IRES-mediated translation from bicistronic reporter constructs, data obtained from two subgenomic HCV replicons and liver tissue do not indicate a significant role of PTB for HCV replication and chronic HCV infection.

Identification of urocortin mRNA antisense transcripts in rat tissue

Urocortin (UCN) has 45% sequence homology with corticotropin releasing factor (CRF) and binds to CRF receptors. We used reverse-transcriptase-polymerase chain reaction to demonstrate the presence of UCN RNA in various brain regions and in peripheral tissues. Ribonuclease protection assay (RPA) using sense and antisense riboprobes demonstrated the presence of a naturally occurring antisense UCN RNA transcript in a number of tissues. Northern blot indicated that the antisense transcript was the same size as sense UCN. RPA, using probes that covered bases 1 to 560 of 579 bp sequence of rat UCN, indicated that the antisense sequence was complementary to sense UCN but did not contain an open reading frame. Sense and antisense UCN RNA were co-expressed in all tissues that contained levels of either transcript detectable by RPA. Sense RNA expression was greater than antisense in the midbrain, the two transcripts were expressed equally in the hypothalamus and antisense was expressed at higher levels than sense in the liver, heart, and skeletal muscle. Antisense RNA expression was stress responsive, suggesting that it may play a role in regulating transcription or translation of UCN mRNA.

Inhibition of eFGF expression in Xenopus embryos by antisense mRNA.

We studied the effects in Xenopus embryos of overexpression of antisense RNA complementary to the messenger RNA of eFGF. We show that the expression of sense RNA can be severely depressed in the presence of an excess of antisense RNA. This occurs by both partial destruction of the message and by a depression of translation of the residual message. The diminution of inducing activity of eFGF, measured in animal cap assays either by activation of the Brachyury gene or by morphology, parallels the reduction of translation. Endogenous eFGF expression is reduced to a similar extent, again by a combination of mRNA destruction and inhibition of translation. This shows that the overexpression of antisense RNA is, contrary to general opinion, a potentially useful technique for studying gene function in Xenopus embryos. However, in the case of eFGF, there is little or no overall phenotypic effect on whole embryos. This is probably because of the presence of several other FGFs with overlapping expression domains in the early embryo.

Stable expression in rat glioma cells of sense and antisense nucleic acids to a human multifunctional DNA repair enzyme, APEX nuclease

We have cloned mouse and human cDNAs for a multifunctional DNA repair enzyme (APEX nuclease) having apurinic/apyrimidinic (AP) endonuclease, 3′–5′ exonuclease, DNA 3′ repair diesterase and DNA 3′-phosphatase activities. To investigate the biological role of APEX nuclease, sense or antisense APEX RNA was stably expressed at a high level in cultured rat glioma cells by introducing plasmids (pABWN-HAPX1F for expression of sense RNA or pABWN-HAPX2R for expression of antisense RNA) constructed from the human APEX cDNA and an expression vector pABWN. Multiple copies of the construct were integrated into the glioma cells tranfected with pABWN-HAPX1F or pABWN-HAPX2R. These transfectants showed markedly high expression of RNA hybridizable to human APEX cDNA, indicating the expression of the sense or antisense RNA. Activity blotting analyses of salt extracts of these transfectants showed that the sense RNA-expressed cells had higher AP endonuclease activity and that the antisense RNA-expressed cells had extremely lower AP endonuclease activity than the control cells. The APEX nuclease-depressed glioma cells became more sensitive to methyl methanesulfonate and hydrogen peroxide than the control cells or the APEX nuclease-overexpressed cells. The results indicate that APEX nuclease plays an important role in repair of DNA damage caused by these genotoxic agents. The present stable expression systems for the sense and antisense APEX RNAs should be useful for analyzing the biological functions such as an antimutagenic function of the enzyme.

Inhibition of human immunodeficiency virus type 1 multiplication by antisense and sense RNA expression

Human immunodeficiency virus type 1 (HIV-1) primarily infects CD4+ lymphocytes and macrophages and causes AIDS in humans. Retroviral vectors allowing neomycin phosphotransferase (npt) gene expression were engineered to express 5' sequences of HIV-1 RNA in the antisense or sense orientation and used to transform the human CD4+ lymphocyte-derived MT4 cell line. Cells expressing antisense or sense RNA to the HIV-1 tat mRNA leader sequence, as part of the 3' untranslated region of the npt mRNA, remained sensitive to HIV-1 infection. In contrast, resistance to HIV-1 infection was observed in cells expressing antisense RNA to the HIV-1 primer-binding site or to the region 5' to the primer-binding site as part of the 3' region of the npt mRNA. Cells expressing the tat mRNA leader sequence in the sense orientation as a precise replacement of the 5' untranslated region of npt mRNA were also resistant to HIV-1. These results indicate that sense and antisense approaches can be used to interfere with HIV-1 multiplication.