Decreased globin messenger RNA in thalassemia by hydridization and biologic activity assays.
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Anemia, Hemolytic↗
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Enhanced degradation of the ferritin repressor protein during induction of ferritin messenger RNA translation.
Induction of ferritin synthesis in cultured cells by heme or iron is accompanied by degradation of the ferritin repressor protein (FRP). Intermediates in the degradative pathway apparently include FRP covalently linked in larger aggregates. The effect of iron on FRP degradation is enhanced by porphyrin precursors but is decreased by inhibitors of porphyrin synthesis, which implies that heme is an active agent. These results suggest that translational induction in this system may be caused by enhanced repressor degradation. While unique among translational regulatory systems, this process is common to a variety of other biosynthetic control mechanisms.
5-Aminolevulinate Synthetase↗
Mechanisms of alternative pre-messenger RNA splicing.
Alternative pre-mRNA splicing is a central mode of genetic regulation in higher eukaryotes. Variability in splicing patterns is a major source of protein diversity from the genome. In this review, I describe what is currently known of the molecular mechanisms that control changes in splice site choice. I start with the best-characterized systems from the Drosophila sex determination pathway, and then describe the regulators of other systems about whose mechanisms there is some data. How these regulators are combined into complex systems of tissue-specific splicing is discussed. In conclusion, very recent studies are presented that point to new directions for understanding alternative splicing and its mechanisms.
Alternative Splicing↗
Cloning and characterization of mouse growth hormone-releasing hormone (GRH) complementary DNA: increased GRH messenger RNA levels in the growth hormone-deficient lit/lit mouse.
We have isolated and cloned the full length cDNA for mouse GH-releasing hormone (mGRH) from mouse hypothalamus using a recently described strategy involving the polymerase chain reaction technique (PCR). Degenerate oligonucleotide primers were selected based on short (six amino acids) conserved regions in the human and rat GRH peptides that would recognize DNA sequences encoding similar amino acids regardless of codon usage. Primer-extended cDNA was amplified by PCR on cDNA templates prepared by reverse transcribing total mouse hypothalamic RNA. After cloning and sequencing the initial product, the 3' and 5' ends of mGRH were generated using a separate PCR strategy (RACE protocol). The mGRH cDNA encodes a 103-amino acid reading frame, structurally similar to the human and rat GRH genes, containing a signal sequence, a 42-residue GRH peptide, and a 31-residue C-terminal region. Although the structures of mouse and rat GRH are highly conserved in the signal peptide and C-terminal region, there is considerable diversity in the GRH region, which exhibits nearly comparable homology with the rat (68%) and human (62%) structures. Differences between mouse and rat GRH were also found in the amino acid cleavage sites at the 5' and 3' ends of the mature peptide and at the polyadenylation signal.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Jansen-type metaphyseal chondrodysplasia: analysis of PTH/PTH-related protein receptor messenger RNA by the reverse transcriptase-polymerase chain method.
Jansen-type metaphyseal chondrodysplasia (JMC) has both delayed ossification in long bones and usually hypercalcemia. We report a Japanese male patient with JMC who presented with rachitic signs on radiographs, hypercalcemia (13 mg/dl) and low %TRP at age 3 months (mo). Hypercalcemia was treated from age 3 mo to 11 yr. Progressive widening, splaying and fragmentation of the metaphyses have been recognized on radiographs which resulted in shortened tubular bones and consequent short stature [107 cm (-6.5 SD)] at age 13 yr. Hypercalcemia tended to normalize, and %TRP became normal at age 13 yr. Repeated measurements of serum PTH and PTH-related protein (PTHrP) levels showed that they were low or normal in the face of hypercalcemia and high urine cAMP excretion, which led us to suspect constitutive activation of the PTH/PTHrP receptor. Direct sequencing of PTH/PTHrP receptor complementary DNA from skin fibroblast cells revealed a CAC to CGC transversion yielding a strictly conserved His223 to Arg substitution found in 90% of DNA fragment in the second transmembrane domain of the receptor. This mutation created a restriction site Sphl (G/CATG/C). Direct sequencing of genomic DNA and also restriction enzyme digestion revealed heterozygous transition. The mutation was absent in the parents with normal phenotype. We conclude that both dysplastic bone lesions and calcium homeostasis are age-dependent in JMC, and that the His223-Arg substitution is the same as that found in four Caucasian patients with a similar phenotype irrespective of the ethnic difference, and that the preferential expression of an abnormal allele of the PTH/PTHrP receptor mRNA in skin fibroblast despite heterogygotic transversion in the genomic DNA suggests the importance of allele expression.
Dwarfism↗
Formation of messenger RNA in animal cells.
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Animals↗
[Complementary DNA synthesis on templates of alphavirus and interferon messenger RNA].
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Alphavirus↗
Human beta-globin messenger RNA. III. Nucleotide sequences derived from complementary DNA.
Sequences of human beta-globin mRNA were determined by analysis of complementary DNA. beta-mRNA was transcribed into double-stranded cDNA by RNA-dependent DNA polymerase. cDNA was cut by restriction endonucleases and the fragments were terminally labeled by means of polynucleotide kinase and [gamma-32P]ATP. After purification, fragments were degraded by snake venom phosphodiesterase. Alternatively single-stranded [32P]cDNA was prepared by transcription in the presence of [alpha-32P]dCTP and actinomycin D; the product was digested by endonuclease IV and degraded by snake venom phosphodiesterase. cDNA tracts obtained by both labeling methods enabled us to construct a sequence for the translated and 3'-terminal untranslated regions of human beta-mRNA.
Anemia, Sickle Cell↗
[Expression of cytokine messenger RNA in mice in physiological conditions].
The expression of the cytokine genes was studied under physiological conditions in normal adult mice using RT-PCR method capable of detecting low levels of mRNA. Total RNA was prepared from brain, spinal cord, lung, spleen, liver and kidney of 6 to 8 week-old specific pathogen-free BALB/c mice by acid guanidinium thiocyanate-phenol-chloroform (AGPC) method. cDNA was synthesized by M-MLV reverse transcriptase, and amplified using the specific oligonucleotide primers for IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, TNF-alpha, IFN-alpha, IFN-beta and IFN-gamma by PCR method. Although IL-1 mRNA was detected in all the organs, IL-3 mRNA was not detected in any organs tested. IL-2 or IL-4 mRNA and IL-5 mRNA were produced only in spleen and lung, respectively. However, IL-6, TNF-alpha and IFN mRNA were detected in some different organs. These results suggest that many kinds of cytokine mRNA were produced in vivo under physiological conditions in normal mice.
Animals↗
Reverse transcription of purified prolactin messenger RNA [proceedings].
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Pituitary Gland↗
Decreasing the distance between the two conserved sequence elements of histone pre-messenger RNA interferes with 3' processing in vitro.
Histone mRNA 3' end formation requires the presence of two cis-acting conserved sequence elements: a stem-loop structure upstream from the site of cleavage and a purine-rich region downstream from the site of cleavage called the histone downstream element (HDE). Possible interactions between these two elements and their respective binding factors were investigated by a series of deletions (1-7 nt) in the region between the two. The efficiency of processing decreased as the stem-loop and the HDE were moved closer together. In contrast with the documented ability of the U7 snRNP to direct cleavage at a fixed distance from the HDE in insertion mutants (Scharl & Steitz, 1994), all deletion substrates for which processing was observed were cleaved at or 1-nt upstream from the wild-type site. The reason for the inability of the system to cleave closer to the stem-loop remains unclear, but the removal of stem-loop binding protein(s) (SLBP) did not activate upstream cleavage events. Thus, although the processing machinery measures the distance between the cleavage site and the HDE of mammalian histone pre-mRNAs, there is a barrier limiting how far upstream cleavage can occur. These data allow a reevaluation of the sites of 3' end processing in known histone pre-mRNAs.
Animals↗
Localization of the prostaglandin F2 alpha receptor messenger RNA and protein in the cynomolgus monkey eye.
PURPOSE: To determine the distribution of the prostaglandin F2 alpha (FP) receptor within the monkey eye. METHODS: The expression and localization of the FP receptor was studied by in situ hybridization and immunohistochemistry. Cryosections of the eye were hybridized with a 35S-labeled FP receptor riboprobe, and paraffin sections were immunostained with polyclonal antibodies against an FP receptor peptide. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on cultured cell populations from the eye. RESULTS: Results of the three methods largely correlated with each other. The highest expression of FP receptor mRNA and protein was found in the corneal, conjunctival, and iridial epithelium, the ciliary muscle, and ciliary processes. Iridial and choroidal melanocytes, the retina, and the optic nerve expressed lower levels of both FP receptor message and protein. Using immunohistochemistry, the FP receptor protein was found in connective tissue fibroblasts, the corneal endothelium, and the vasculature; however, FP receptor expression was not detected using in situ hybridization. With RT-PCR, cultured retinal pigment epithelial and ciliary muscle cells from the cynomolgus monkey eye were found to express the FP receptor. CONCLUSIONS: The FP receptor was found to be distributed widely in the ocular tissues, suggesting an array of autocrine and paracrine functions of PGF2 alpha in the eye.
Amino Acid Sequence↗
Probing the structure of the regulatory region of human transferrin receptor messenger RNA and its interaction with iron regulatory protein-1.
A portion of the 3'UTR of the human transferrin receptor mRNA mediates iron-dependent regulation of mRNA stability. The minimal RNA regulatory region contains three conserved hairpins, so-called iron responsive elements (IREs), that are recognized specifically by iron regulatory proteins (IRPs). The structure of this regulatory region and its complex with IRP-1 was probed using a combination of enzymes and chemicals. The data support the existence of an intrinsic IRE loop structure that is constrained by an internal C-G base pair. This particular structure is one of the determinants required for optimal IRP binding. IRP-1 covers one helical turn of the IRE and protects conserved residues in each of the three IREs: the bulged cytosine and nucleotides in the hairpin loops. Two essential IRP-phosphate contacts were identified by ethylation interference. Three-dimensional modeling of one IRE reveals that IRP-1 contacts several bases and the ribose-phosphate backbone located on one face in the deep groove, but contacts also exist with the shallow groove. A conformational change of the IRE loop mediated by IRP-1 binding was visualized by Pb2+-catalyzed hydrolysis. This effect is dependent on the loop structure and on the nature of the closing base pair. Within the regulatory region of transferrin receptor mRNA, IRP-1 induces reactivity changes in a U-rich hairpin loop that requires the presence of the stem-loop structure located just downstream the endonucleolytic cleavage site identified by Binder et al. (Binder R et al. 1994, EMBO J 13:1969-1980). These results provide indications of the mechanism by which IRP-1 stabilizes the transferrin receptor mRNA under iron depletion conditions.
Base Composition↗
Posttranscriptional regulation of protein expression in human epithelial carcinoma cells by adenine-uridine-rich elements in the 3'-untranslated region of tumor necrosis factor-alpha messenger RNA.
Eukaryotic mRNAs contain 3'-untranslated regions (UTR) that are involved in posttranscriptional control of gene expression. AU-rich octanucleotide repeats, UUAUUUAU, present in the 3'-UTR of mature lymphokine and other cytokine transcripts, have been implicated in the regulation of mRNA stability and translational efficiency. For example, previous evidence suggests that the AU-rich element (ARE) present in the 3'-UTR of murine tumor necrosis factor-alpha (TNF-alpha) can affect the posttranscriptional regulation of murine TNF-alpha gene expression in hematopoietic cells. Although cytokines are produced in epithelial cells, little is known about the regulation of TNF-alpha and other cytokine gene expression by 3'-UTR elements in human malignant epithelial cells. To better understand the function of the 3'-UTR of the human TNF-alpha gene in the regulation of TNF-alpha protein production in human epithelial cancer cells, a series of luciferase reporter constructs with portions of the 3'-UTR of human TNF-alpha was transfected into human breast carcinoma cell lines ZR-75-1 and ZR-75-1R (which overexpresses TNF-alpha). The 3'-UTR of TNF-alpha markedly suppressed luciferase activity in both cell lines, and the suppression of activity was reversed by deletion of the AU-rich sequences. This suppression was quantitative, with six repeats causing more inhibition than two repeats. Increased levels of luciferase activity were observed 3 h after TNF-alpha stimulation in ZR-75-1 cells transfected by constructs containing AU-rich repeats. In addition, cytoplasmic extracts from both cell lines were assayed for factors that bind to the 3'-UTR of human TNF-alpha mRNA. RNA-protein binding activities were found in both cell lines. Competition studies showed that these proteins specifically bound to AU-rich repeats present in the 3'-UTR of TNF-alpha. No binding activity was observed when the AU-rich repeats were deleted. TNF-alpha exposure markedly increased activity of several RNA-binding proteins, especially a novel Mr 50,000-55,000 RNA-binding protein. The binding activity in untreated ZR-75-1R was higher than that in untreated ZR-75-1 cells, suggesting that the level of RNA-protein binding correlates with the expression level of TNF-alpha in human epithelial cancer cells and that the RNA-binding proteins may control expression of TNF-alpha in ZR-75-1 cells. We conclude that the AU-rich repeats in the 3'-UTR of human TNF-alpha mRNA may regulate gene expression in human epithelial cancer cells by binding to AU sequence-specific proteins, including a previously undescribed Mr 50,000-55,000 protein not observed in hematopoietic cells.
Animals↗
Exfoliated oral cell messenger RNA: suitability for biomarker studies.
The efficacy of chemoprevention trials can be improved by the use of biomarkers of carcinogenesis that serve as surrogate end points. The aim of this study was to assess the perspectives of using mRNA isolated from oral exfoliated cells for biomarker research in chemoprevention of upper aerodigestive tract cancer. When using reverse transcription-PCR in combination with Southern blotting and hybridization, it was possible to detect transcripts from only five cells. With the quantitative RNase protection assay, we could only detect highly abundant transcripts. The integrity of the RNA was verified by Northern blotting, which showed a variable degree of degradation, depending on the gene studied. Interestingly, although specific transcripts were found to be intact to a certain extent, the rRNA appeared to be completely degraded, suggesting that a specific protein synthesis shut-off mechanism exists in terminally differentiated oral epithelial cells. Altogether, this differential RNA degradation makes accurate measurement of transcript levels of most genes, as determined in exfoliated oral cells, unreliable. Because this RNA degradation process is likely to start before the cells are shed from the tissue, the results of measurements of transcript levels in biopsies of oral tissue should be interpreted with caution.
Adult↗
Glutamate decarboxylase messenger RNA in rat pallidum: comparison of the effects of haloperidol, clozapine and combined haloperidol-scopolamine treatments.
We have investigated the effects of neuroleptic treatments which do, or do not, induce catalepsy on the level of expression of glutamate decarboxylase, the rate limiting enzyme in GABA synthesis, in efferent neurons of the pallidum in adult rats. Different regimens of haloperidol (1 mg/kg s.c., three, seven or 14 days; 2 mg/kg, s.c., 10 days) induced catalepsy in a majority of rats and increased glutamate decarboxylase messenger RNA levels in the globus pallidus (external pallidum) in those rats exhibiting catalepsy. Levels of glutamate decarboxylase messenger RNA were also increased in the entopeduncular nucleus (internal pallidum), but only after 14 days of treatment with haloperidol. The atypical antipsychotic clozapine (seven days, 20 mg/kg, s.c.), which did not induce catalepsy, slightly decreased glutamate decarboxylase messenger RNA levels in the globus pallidus. When co-administered with haloperidol (seven days, 1 mg/kg s.c.), the muscarinic antagonist scopolamine (1 mg/kg, s.c.) completely blocked both haloperidol-induced catalepsy and increases in glutamate decarboxylase messenger RNA levels in the globus pallidus. In contrast, scopolamine was not able to block increased glutamate decarboxylase and enkephalin messenger RNA expression induced by haloperidol in the striatum. These results reveal a good correlation between increases in glutamate decarboxylase messenger RNA levels in the globus pallidus and catalepsy after these drug treatments and suggest that anticholinergic blockade of the behavioral and molecular effects of neuroleptics may involve non-striatal mechanisms.
Animals↗
Engineered recombinant messenger RNA can be replicated and expressed inside bacterial cells by an RNA bacteriophage replicase.
In this paper we describe how an RNA replicase can be "tricked" into recognizing, binding to, and replicating host-cell message RNAs. In our system, the gene encoding Q beta replicase is constitutively expressed from a plasmid vector present in an Escherichia coli host, while a second plasmid directs the transcription of a replication-competent, phage-like template RNA. This 680 nucleotide transcript (N- RNA) contains specific sequences required for Q beta replicase function. Included within this template RNA is a 360-nucleotide sequence that is complementary to the messenger RNA for DNA bacteriophage lambda N protein. The active messenger RNA for lambda N protein is expressed only upon replication of N- RNA by Q beta replicase. By employing an E. coli host strain that requires lambda N protein for growth under specific selective conditions, we were able to select only those cells in which RNA-directed RNA replication occurred. This system provides a potential for in vivo amplification of other heterologous messenger RNAs and their protein products via RNA replication and will enable one to study the evolution of messenger RNA in the live cell under a large variety of physiological pressures.
Base Sequence↗
Enhanced expression of metabotropic glutamate receptor 3 messenger RNA in the rat spinal cord during ultraviolet irradiation induced peripheral inflammation.
Metabotropic glutamate receptors are thought to play a role in the development and maintenance of spinal hyperexcitability resulting in hyperalgesia and pain. In this study we have used in situ hybridization to investigate the distribution of metabotropic glutamate receptors mGluR1-7 messenger RNA in the rat spinal cord in a model of inflammatory hyperalgesia. Hyperalgesia was induced in nine-day-old rats by exposure of the left hindpaw to an ultraviolet light source. Lumbar portions of spinal cords were removed from control and ultraviolet-treated animals. In situ hybridization with specific oligonucleotide probes was used to localize metabotropic glutamate receptor messenger RNAs. mGluR1, 3-5 and 7 subtype messenger RNA was detected in the gray matter of the spinal cord with distribution being specific for the different subtypes. A significant increase in the expression of mGluR3 messenger RNA was seen in cells of the dorsal laminae in both sides of the lumbar spinal cord. This increase was most pronounced in laminae II, III and IV but gradually decreased and disappeared by the third day of inflammation. In parallel with this, behavioural experiments revealed mechanical hyperalgesia in both hindlimbs after ultraviolet irradiation. There was no change in mGluR3 messenger RNA expression in the thoracic segments. No changes have been detected in the levels of expression of mGluR 1,2,4,5,7 subtype messenger RNA in spinal cords taken from hyperalgesic animals. These observations show that during ultraviolet irradiation induced inflammation, the synthesis of mGluR3 messenger RNA is altered suggesting that regulation of metabotropic glutamate receptor expression may be instrumental in plastic changes within the spinal cord during the development of hyperalgesia and pain.
Amino Acid Sequence↗