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We evaluated the expression of over 900 AU-rich element (ARE)-containing transcripts in primary human T lymphocytes following stimulation with anti-CD3 and anti-CD28 antibodies and found that approximately 48% of these transcripts were regulated following T cell activation. We identified approximately 145 ARE-containing transcripts that were rapidly induced and then rapidly disappeared within 1 h after activation. Another 250 ARE-containing transcripts expressed in resting T cells were rapidly turned off within 30 min after activation. The rates of transcript disappearance correlated well with rapid mRNA decay measured following transcriptional arrest with actinomycin D. We identified a subset of ARE-containing transcripts that were rapidly induced following T cell activation that were also induced following lipopolysaccharide stimulation of THP-1 monocytes, and these transcripts exhibited rapid decay in both cell types. Our results suggest that ARE-mediated mRNA decay plays an important role in the precisely coordinated down-regulation of gene expression following immune cell activation.
CPSI deficiency is an inborn error of metabolism caused by mutations in the first, rate-determining enzyme of the urea cycle. Our mutation detection data from this disorder suggest that a significant number of mutant alleles cause RNA instability, most likely through the nonsense-mediated decay pathway. We identified 26 non-consanguinous CPSID patients with an available RNA source (liver tissue or cell line) and screened both genomic DNA and RNA for the identification and classification of mutations. Out of 52 total alleles screened from these patients, 21 (40%) have strong evidence for RNA processing mutations demonstrated by absent/minimal heterozygosity in patient cDNA sequences despite heterozygous genomic changes. These 21 alleles are a heterogenous group primarily composed of splicing defects and frameshifts that form premature termination codons which should subsequently elicit the nonsense-mediated decay pathway. This study provides evidence for the high prevalence of RNA instability mutations in genetic disease and underscores the importance of accounting for them in mutation-screening strategies.
Succinyl-CoA:3-ketoacid CoA transferase (SCOT, EC 2.8.3.5) is the key enzyme for ketone body utilization. Hereditary SCOT deficiency (MIM 245050) causes episodes of severe ketoacidosis. We identified a homozygous point mutation (c.671G>A) , which is a single-base substitution at the last nucleotide of exon 6, in a Turkish patient (GS12) with SCOT deficiency. This point mutation resulted in the skipping of exon 6, and exons 6 and 7 in human SCOT genes. To understand why the c.671G>A causes exons 6 and 7 skipping, nuclear RNA was separated from cytoplasmic RNA and both were analyzed by RT-PCR. In nuclear RNA, SCOT mRNA with exon 6 skipping was predominant and mRNA with exons 6 and 7 skipping was hardly detected, whereas the latter became one of major mRNA species in cytoplasmic RNA. This discrepancy was interpreted as follows: exon 6 skipping causes a frameshift and nonsense-mediated RNA decay in the cytosol, so mRNA with exon 6 skipping was unstable. On the other hand, SCOT mRNA with exons 6 and 7 is a minor transcript but it retains the reading-frame and is stable in cytosol. As a result, the latter mRNA is more abundant under steady-state conditions as compared to the former mRNA.
Nonsense-mediated mRNA decay (NMD) is an mRNA quality-control mechanism that degrades aberrant mRNAs containing premature translation termination codons (PTCs). The essential proteins for NMD include SMG-1, a protein kinase, and Upf1, a substrate of SMG-1 with RNA helicase activity. In this study, we evaluated the effects of NMD inhibition by siRNA-mediated knockdown of SMG-1 or Upf1 on the phenotype of Ullrich disease, an autosomal recessive congenital muscular dystrophy. The patient studied showed a homozygous frameshift mutation with a PTC in the collagen VI alpha2 gene, which encodes a truncated but partially functional protein. The patient's fibroblasts showed a nearly complete loss of the triple-helical collagen VI protein and functional defects in the extracellular matrix (ECM) due to the crucial deficiency of the collagen VI alpha2 protein. We have shown that siRNA-mediated knockdown of SMG-1 or Upf1 causes the up-regulation of the mutant triple-helical collagen VI, resulting in the formation of partially functional ECM. We suggest that the inhibition of NMD may be useful as a therapeutic approach to treat some human genetic diseases exacerbated by NMD.
Apoptosis could be measured in mammalian cells by measuring the degradation of the small cytoplasmic human RNA Y1 (hY1) by real-time quantitative fluorescent reverse transcriptase polymerase chain reaction (RT-PCR). In FAS-antibody-treated Jurkat T cell leukemia cells degradation of hY1 occurred rapidly and was complete at about 6h. As in apoptotic Jurkat cells, protein synthesis is arrested only after about 12h; this implies that protein synthesis can occur without scRNA-Y1. The degradation of hY1 could be blocked with peptide-based inhibitors of caspase 8 and with lower efficacy with caspases 1 and 3 and with ZnSO4. No effects were observed after inhibition of caspases 2, 6, and 9. Degradation of hY1 could also be demonstrated after treatment of A549 lung carcinoma cells treated with Staurosporin, Paclitaxel, or the histone deacetylase inhibitor LAQ824. RT-PCR systems based on SYBR Green, Amplifluor Uniprimer, or 5' nuclease Taqman could be used with increasing sensitivity. This apoptosis assay requires quantities of total cell RNA equivalent to only a few tissue culture cells and is especially suited to measure apoptosis in projects where RNA samples are already available from gene expression studies.
High-glucose exposure down-regulates protein kinaseC beta II posttranscriptionally in rat and human vascular smooth muscle cells and contributes to increased cell proliferation. High-glucose-induced mRNA destabilization is specific for PKC beta II mRNA, while PKC beta I and other PKC mRNA are not affected. This study focused on whether glucose metabolism was required. The effect was blocked by cytochalasin B, suggesting a requirement for glucose uptake. Glucosamine did not mimic the effect, indicating that metabolism via hexosamine pathway was not involved. The effect was hexokinase-independent since 3-O-methylglucose, in a dose-dependent manner, mimicked high-glucose effects. Cycloheximide did not block the effect excluding dependency on new protein synthesis. Wortmannin and LY294002, phosphoinositide 3-kinase (PI3-kinase) inhibitors, blocked glucose effects in the presence of 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole. Glucose and 3-O-methylglucose activated PI3-kinase, and LY294002 blocked glucose effects on Akt phosphorylation. In these cells, high-glucose concentrations activated a metabolically linked signaling pathway independent of glucose metabolism to regulate mRNA processing.
Hepatitis B virus (HBV) X protein (HBx) is thought to be involved in the development of liver cancer and alteration of cellular HBx level may influence the pathological progression of HBV-induced liver diseases. We found that the cellular levels of HBx mRNA transcript and protein in cells were greatly enhanced by adriamycin, a topoisomerase II inhibitor. Up-regulation of HBx mRNA by adriamycin was also observed in HBx transgenic mice, which was accompanied with a significant increase of VEGF mRNA, the downstream target of HBx. When we investigated the underlying mechanism, we found that half-life of HBx mRNA in HBx-expressing Chang cells was about 3h, but was prolonged to >6h in the presence of adriamycin. Moreover, half-life of rapidly degrading HBx protein was determined as about 15min however, it remained almost constant until 60min in the presence of adriamycin. These results provide the first evidence that the cellular level of HBx gene can be increased at the post-transcriptional level.
Double-stranded RNA (dsRNA) homologous to the Toxoplasma gondii uracil phosphoribosyltransferase (TgUPRT) gene is able to modulate the UPRT gene expression in T. gondii. The dsRNA, which was produced either from a constructed plasmid or from an in vitro transcription reaction, was capable of down-regulating the expression of TgUPRT. Stably transformed T. gondii expressing the dsRNA, which was capable of growing in the presence of the prodrug 5-fluoro-2(')-deoxyuridine (FDUR), appeared to maintain the engineered plasmid as an extra-chromosomal DNA. When cultured in the absence of the selection pressure, the FDUR resistant parasites slowly reverted to the FDUR sensitive phenotype. The level of the dsRNA necessary to confer FDUR resistance was estimated at 2-8 copies per parasite. More importantly the introduction of the in vitro synthesized dsRNA homologous to the TgUPRT gene into T. gondii can also induce the specific mRNA degradation, resulting in a lowered UPRT activity.
alpha(1)-Acid glycoprotein (AGP) is the major transport protein for cationic drugs, endogenous ligands, and some anionic drugs in plasma. Hepatic synthesis and secretion of AGP are altered during acute inflammation as well as by a number of drugs. This alteration could influence the binding of drugs and its biological function. Macrolide antibiotics are widely used in the treatment of a variety of infectious diseases. The effects of macrolide antibiotics have been studied with respect to rat AGP expression in vivo. After the individual administration of six macrolides to rats, with the exception of oleandomycin, five increased AGP levels in serum. Of these five, clarithromycin (CAM) was the most potent inducer of AGP, which reached a maximum level between 3 to 7 days after administration. CAM increased the steady-state level of AGP mRNA in liver as well as protein level in serum in a dose-dependent manner. In addition, CAM increased AGP mRNA levels in primary cultured hepatocytes. In the luciferase promoter assay, CAM potentiated dexamethasone-increased promoter activity of the AGP gene, which contained the glucocorticoid response element, in cultured rat hepatocytes, although CAM itself had no effect on its activity. The effect of CAM and dexamethasone was diminished by glucocorticoid response element deletion or mutation or by adding the antiglucocorticoid, RU486. Further, in the mouse mammary tumor virus (MMTV) promoter containing functional glucocorticoid response element, CAM potentiated dexamethasone-increased promoter activity. In the adrenalectomized rats, CAM did not increase AGP levels in serum. These findings suggest that CAM may cause transcriptional induction of AGP, at least in part, via a glucocorticoid-mediated mechanism.
We have analyzed the steady-state levels of cytochrome P-450 1A1 (CYP1A1) mRNA in peripheral blood lymphocytes of 177 individuals with various CYP1A1 genotypes using a quantitative reverse transcriptase-polymerase chain reaction technique that makes use of a homologous internal standard for accurate quantitation. We found no effects of ethnicity, age, or smoking status on CYP1A1 gene expression in this population. We did see a significant 2-fold increase in the mean level of CYP1A1 mRNA in women compared with men for both Caucasians and African Americans. We observed no effect of the African American-specific polymorphism (CYP1A1(*)3) on expression of the gene. However, we found a significant 3-fold decrease in expression associated with the homozygous MspI restriction fragment length polymorphism (CYP1A1(*)2A/(*)2A).
Mitochondrial (mt) transfer RNAs (tRNAs) often harbor unusual structural features causing their secondary structure to differ from the conventional cloverleaf. tRNAs designed with such irregularities, termed mt-like tRNAs, are active in Escherichia coli as suppressors of reporter genes, although they display low steady-state levels. Characterization of fragments produced during mt-like tRNA processing in vitro and in vivo suggests that these RNAs are not fully processed at their 5' ends and are cleaved internally. These abnormal processing events may account for the low levels of mature mt-like RNAs in vivo and are most likely related to defective processing by RNase P.
Scorpion venom glands synthesize and secrete a great number of low molecular mass toxic peptides for prey and defense. Many cDNAs and genomic genes encoding these toxins have been isolated and sequenced. However, their expression regulation mechanism is not yet known at present. During screening of a cDNA library prepared from venom glands of the scorpion Buthus martensii Karsch, we isolated a natural fusion cDNA composed of the 5'-untranslated region (UTR) and upstream coding sequence of a long-chain toxin transcript and the downstream coding sequence and 3'-UTR of a short-chain toxin transcript. The junction site is just the overlapping region of 11 nucleotides (GGCAAGGAAAT) between the two wild transcripts, and thus leads to the formation of an early stop codon, which will cause premature translation. Based on the above observations, combined with the genomic data, we proposed a characteristic regulation mechanism of scorpion toxin genes, in which trans-splicing and nonsense mediated mRNA decay are involved.
A cDNA clone up-regulated in hydraulic lung edema in rabbit showed high similarity with human RDH10 mRNA, which encodes a protein involved in retinoic acid metabolism. We defined the organization of the human gene, which includes a unique transcriptional start site, a coding region with six translated exons and a 3' untranslated region containing at least two used polyadenylation sites. The two poly(A) signals are responsible for the production of the 3 and 4 kb RDH10 mRNA isoforms detected in several human tissues and cell lines.
The quantification of the cytokine mRNAs synthesised by peripheral blood cells should make it possible to estimate a "peripheral immune statute". However, an accurate quantification can only be performed from a fresh whole blood sample in which mRNA is protected against nuclease digestion, and where gene transcription is inhibited. As discussed in this note, this has been made possible by the use of surfactant reagents such as tetradecyltrimethylammonium oxalate. We performed RT-PCR for the quantification of IL-10 and IFN-gamma mRNAs spontaneously produced in peripheral blood. The results showed pronounced higher IFN-gamma transcript levels in whole blood compared to peripheral blood mononuclear cells (PBMC) from the same individuals, while no significant difference was observed for IL-10 mRNA. The higher amounts of IFN-gamma mRNA observed in blood can be attributed at least to mRNA degradation. Using a real time PCR technique, we indeed demonstrated that blood IFN-gamma mRNA is rapidly degraded in vitro, the t 1/2 being worth approximately one hour at room temperature.
The folding pathway of the Tetrahymena ribozyme correlates inversely with the sequence distance between native interactions, or contact order. The rapidly folding P4-P6 domain has a low contact order, while the slowly folding P3-P7 region has a high contact order. To examine the role of topology and contact order in RNA folding, we screened for circular permutants of the ribozyme that retain catalytic activity. Permutants beginning in the P4-P6 domain fold 5 to 20 times more slowly than the wild-type ribozyme. By contrast, 50% of a permuted RNA that disjoins a non-native interaction in P3 folds tenfold faster than the wild-type ribozyme. Hence, the probability of rapidly folding to the native state depends on the topology of tertiary domains.
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