• Tuberculosis and Respiratory Diseases
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• 제56권4호
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• pp.393-404
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• 2004

Background : Nucleic acid hybridization has become an essential technique in the development of our understanding of gene structure and function. The quantitative analysis of hybridization has been used in the measurement of genome complexity and gene copy number. The filter hybridization assay is rapid, sensitive and can be used to measure RNAs complementary to any cloned DNA sequence. Methods : The authors assessed the accuracy, linearity, correlation coefficient and specificity of the hybridization depending on the added dose(0, 1, 5, and $10{\mu}g$) of non-specific rat spleen RNA to hybridization of surfactant protein A mRNA. Filter hybridization assays were used to obtain the equation of standard curve and thereby to quantitate the mRNA quantitation. Results : 1. Standard curve equation of filter hybridization assay between counts per minute (X) and spleen RNA input (Y) was Y=0.13X-19.35. Correlation coefficient was 0.98. 2. Standard curve equation of filter hybridization assay between counts per minute (X) and surfactant protein A mRNA transcript input (Y) was Y=0.00066X-0.046. Correlation coefficient was 0.99. 3. Standard curve equation of filter hybridization assay between counts per minute (X) and surfactant protein A mRNA transcript input (Y) after the addition of $1{\mu}g$ spleen RNA was Y=0.00056X-0.051. Correlation coefficient was 0.99. 4. Standard curve equation of filter hybridization assay between counts per minute (X) and surfactant protein A mRNA transcript input (Y) after the addition of $5{\mu}g$ spleen RNA was Y=0.00065X-0.088. Correlation coefficient was 0.99. 5. Standard curve equation of filter hybridization assay between counts per minute (X) and surfactant protein A mRNA transcript input (Y) after the addition of $10{\mu}g$ spleen RNA was Y=0.00051X-0.10. Correlation coefficient was 0.99. Conclusions : Comparison of cpm/filter in a linear range allowed accurate and reproducible estimation of surfactant protein A mRNA copy number irrespective of the addition dosage of non-specific rat spleen RNA over the range $0-10{\mu}g$.

• Journal of Microbiology and Biotechnology
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• 제24권2호
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• pp.287-294
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• 2014

The transcription factor E2F1 is active during G1 to S transition and is involved in the cell cycle and progression. A recent study reported that increased E2F1 is associated with DNA damage and tumor development in several tissues using transgenic models. Here, we show that E2F1 expression is regulated by tristetraprolin (TTP) in prostate cancer. Overexpression of TTP decreased the stability of E2F1 mRNA and the expression level of E2F1. In contrast, inhibition of TTP using siRNA increased the E2F1 expression. E2F1 mRNA contains three AREs within the 3'UTR, and TTP destabilized a luciferase mRNA that contained the E2F1 mRNA 3'UTR. Analyses of point mutants of the E2F1 mRNA 3'UTR demonstrated that ARE2 was mostly responsible for the TTP-mediated destabilization of E2F1 mRNA. RNA EMSA revealed that TTP binds directly to the E2F1 mRNA 3'UTR of ARE2. Moreover, treatment with siRNA against TTP increased the proliferation of PC3 human prostate cancer cells. Taken together, these results demonstrate that E2F1 mRNA is a physiological target of TTP and suggests that TTP controls proliferation as well as migration and invasion through the regulation of E2F1 mRNA stability.

• The Korean Journal of Physiology and Pharmacology
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• 제23권3호
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• pp.181-189
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• 2019

Curcumin, an active ingredient of Curcuma longa L., can reduce the concentration of low-density lipoproteins in plasma, in different ways. We had first reported that curcumin exhibits hypocholesterolemic properties by improving the apolipoprotein B (apoB) mRNA editing in primary rat hepatocytes. However, the role of curcumin in the regulation of apoB mRNA editing is not clear. Thus, we investigated the effect of curcumin on the expression of multiple editing components of apoB mRNA cytidine deamination to uridine (C-to-U) editosome. Our results demonstrated that treatment with $50{\mu}M$ curcumin markedly increased the amount of edited apoB mRNA in primary mouse hepatocytes from 5.13%-8.05% to 27.63%-35.61%, and significantly elevated the levels of the core components apoB editing catalytic polypeptide-1 (APOBEC-1), apobec-1 complementation factor (ACF), and RNA-binding-motif-protein-47 (RBM47), as well as suppressed the level of the inhibitory component glycine-arginine-tyrosine-rich RNA binding protein. Moreover, the increased apoB RNA editing by $50{\mu}M$ curcumin was significantly reduced by siRNA-mediated APOBEC-1, ACF, and RBM47 knockdown. These findings suggest that curcumin modulates apoB mRNA editing by coordinating the multiple editing components of the edito-some in primary hepatocytes. Our data provided evidence for curcumin to be used therapeutically to prevent atherosclerosis.

• BMB Reports
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• 제48권7호
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• pp.367-368
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• 2015

It has long been thought that glucocorticoid receptor (GR) functions as a DNA-binding transcription factor in response to its ligand (a glucocorticoid) and thus regulates various cellular and physiological processes. It is also known that GR can bind not only to DNA but also to mRNA; this observation points to the possible role of GR in mRNA metabolism. Recent data revealed a molecular mechanism by which binding of GR to target mRNA elicits rapid mRNA degradation. GR binds to specific RNA sequences regardless of the presence of a ligand. In the presence of a ligand, however, the mRNA-associated GR can recruit PNRC2 and UPF1, both of which are specific factors involved in nonsense-mediated mRNA decay (NMD). PNRC2 then recruits the decapping complex, consequently promoting mRNA degradation. This mode of mRNA decay is termed "GR-mediated mRNA decay" (GMD). Further research demonstrated that GMD plays a critical role in chemotaxis of immune cells by targeting CCL2 mRNA. All these observations provide molecular insights into a previously unappreciated function of GR in posttranscriptional regulation of gene expression. [BMB Reports 2015; 48(7): 367-368]

• Asian-Australasian Journal of Animal Sciences
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• 제18권5호
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• pp.734-740
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• 2005

The blood samples were collected from dairy cows at the same milking stage. The single-strand conformation polymorphism (PCR-SSCP) method was used to analyze for polymorphism at the 5'flanking region of the hsp70 gene. The mRNA expression levels of HSP70, HSF1, Bcl-2 and Bax-$\alpha$ at different daily-mean-temperature were analyzed by relative quantitative RTPCR. The DNA content, cell phase and the ratio of apoptosis of lymphocytes in peripheral blood of dairy cattle at different daily-meantemperature were determined by FCM. The PCR-SSCP products of primer pair 1 showed polymorphisms and could be divided into four genotypes: aa, ab, ac, cc, with the cis-acting element (CCAAT box) included. Mutations in the hsp70 5'flanking region (468-752 bp) had different effects on mRNA expression of HSP70, HSF1, Bcl-2 and Bax-$\alpha$. The ac genotypic cows showed higher expressions of HSP70mRNA, HSF1mRNA and Bcl-2mRNA/Bax-$\alpha$mRNA and lower ratio of apoptosis. These mutation sites can be used as molecular genetic markers to assist selection for anti-heat stress cows.

• Molecules and Cells
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• 제37권1호
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• pp.1-8
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• 2014

Mammalian-cell messenger RNAs (mRNAs) are generated in the nucleus from precursor RNAs (pre-mRNAs, which often contain one or more introns) that are complexed with an array of incompletely inventoried proteins. During their biogenesis, pre-mRNAs and their derivative mRNAs are subject to extensive cis-modifications. These modifications promote the binding of distinct polypeptides that mediate a diverse array of functions needed for mRNA metabolism, including nuclear export, inspection by the nonsense-mediated mRNA decay (NMD) quality-control machinery, and synthesis of the encoded protein product. Ribonucleoprotein complex (RNP) remodeling through the loss and gain of protein constituents before and after pre-mRNA splicing, during mRNA export, and within the cytoplasm facilitates NMD, ensuring integrity of the transcriptome. Here we review the mRNP rearrangements that culminate in detection and elimination of faulty transcripts by mammalian-cell NMD.

• Molecules and Cells
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• 제46권11호
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• pp.664-671
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• 2023

The proper maintenance of mRNA quality that is regulated by diverse surveillance pathways is essential for cellular homeostasis and is highly conserved among eukaryotes. Here, we review findings regarding the role of mRNA quality control in the aging and longevity of Caenorhabditis elegans, an outstanding model for aging research. We discuss the recently discovered functions of the proper regulation of nonsense-mediated mRNA decay, ribosome-associated quality control, and mRNA splicing in the aging of C. elegans. We describe how mRNA quality control contributes to longevity conferred by various regimens, including inhibition of insulin/insulin-like growth factor 1 (IGF-1) signaling, dietary restriction, and reduced mechanistic target of rapamycin signaling. This review provides valuable information regarding the relationship between the mRNA quality control and aging in C. elegans, which may lead to insights into healthy longevity in complex organisms, including humans.

• Journal of the Korean Chemical Society
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• 제52권5호
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• pp.580-582
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• 2008

• Journal of Embryo Transfer
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• 제29권3호
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• pp.297-303
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• 2014

The objective of this study was to determine the breed differences in the 3'-untranslated region (UTR) of MC1R mRNA, which may be used to distinguish Korean brindle cattle (Chikso) from other breeds. We investigated the relationship between the variation of 3'-UTR of the MC1R mRNA and coat color among different breeds and the Korean brindle cattle with different coat colors. MC1R mRNA expression levels were determined in accordance with the coat color and hair colors of the tail. Total cellular RNA was extracted from the hair follicles of the tails in Hanwoo, Korean brindle cattle, Holstein and $Hanwoo{\times}Holstein$ crossbred cattle. After cDNA synthesis, PCR was performed. Sequences of the 3'-UTR of MC1R mRNA were analyzed. The 3'-UTR of the MC1R mRNA from different breeds of cattle did not show any variations. There were no variations in the 3'-UTR of the MC1R mRNA in Korean brindle cattle with different coat colors. The levels of MC1R mRNA expression in hair follicles of the tail varied substantially among the Korean brindle cattle with different coat colors, except yellow coat color. Correlation between the MC1R mRNA expression in the hair follicles of the tail and coat color may be present in the Korean brindle cattle, but not between the variations of 3'-UTR of MC1R mRNA and coat color. Further studies to determine the regulation of MC1R mRNA expression from the hair follicles of different coat colors will be beneficial in clarifying the role of MC1R in the coat colors of the Korean brindle cattle.

• Animal cells and systems
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• 제2권4호
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• pp.483-488
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• 1998

We previously found that a potent gonadotropin-releasing hormone (GnRH) agonist, buserelin, decreases GnRH promoter activity together with GnRH mRNA level, providing evidence for an autoregulatory mechanism operating at the level of GnRH gene transcription in immortalized GT1-1 neuronal cells. To examine whether agonist-induced decrease in GnRH mRNA level requires the continuous presence of buserelin, we performed a pulse-chase experiment of buserelin treatment. Short-term exposure (15 min) of GT1-1 neuronal cells to buserelin ($10{\mu}M$) was able to decrease GnRH mRNA levels when determined 24 h later. When GT1-1 cells were treated with buserelin ( $10{\mu}M$) for 30 min and then incubated for 1, 3, 6, 12, 24, and 48 h after buserelin removal, a significant decrease in GnRH mRNA levels was observed after the 12 h incubation period. These data indicate that inhibitory signaling upon buserelin treatment may occur rapidly, but requires a long time (at least 12 h) to significantly decrease the GnRH mRNA level. To examine the possible involvement of de novo synthesis and/or mRNA stability in buserelin-induced decrease in GnRH gene expression, actinomycin D ($5{\mu}m/ml$), a potent RNA synthesis blocker, was co-treated with buserelin. Actinomycin D alone failed to alter basal GnRH mRNA Revel, but blocked the buserelin-induced decrease in GnRH mRNA level at 12 h of post-treatment. These data suggest that buserelin may exert its inhibitory action by altering the stability of GnRH mRNA. Moreover, a polvsomal RNA separation by sucrose gradient centrifugation demonstrated that buserelin decreased the translational efficiency of the transcribed GnRH mRNA. Taken together, these results clearly indicate that GnRH agonist buserelin acts as an inhibitory signal at multiple levels such as transcription mRNA stability, and translation.