Translation. Dissecting RNA function.
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Protein Biosynthesis↗
Biomedical subjects
R Schroeder
Publications and source records attributed to R Schroeder.
Peptide antibiotics of the tuberactinomycin family as inhibitors of group I intron RNA splicing.
The tuberactinomycins are a group of cyclic peptide antibiotics, which are potent inhibitors of prokaryotic protein synthesis. We report the inhibitory effect of viomycin, di-beta-lysyl-capreomycin IIA and tuberactinomycin A on group I intron self-splicing. They compete with the guanosine cofactor for the G-binding site located in the conserved core of the intron. They are 100-fold more active than all other competitive inhibitors described so far (dGTP, arginine or streptomycin), inhibiting splicing at concentrations between 10 and 50 microM. Mutation of the G-binding site leads to partial resistance, and the inhibitory effect of these drugs is dependent on Mg2+ concentration. This suggests that the tuberactinomycins have more than one contact site with the intron RNA: via the G-binding site and via additional contacts with the RNA backbone. Positioning the tuberactinomycins in the three-dimensional model of the td intron core suggests that the charged lysyl side-chain (R1) is in contact with the backbone of the P1 helix. Structure/function analyses with various tuberactinomycin analogues with different activities confirm the involvement of this sidechain in inhibition of group I self-splicing. The demonstration of a new class of splicing inhibitors, the peptide antibiotics, illustrates how antibiotics may interact with catalytic RNA.
Bacteriophage T4↗
Behavioral effects of persistent lymphocytic choriomeningitis virus infection in mice.
Lymphocytic choriomeningitis virus (LCMV) is a nonlytic murine virus that provides a valuable model system for studying the behavioral correlates of CNS viral infection. Newborn or immunosuppressed mice infected with LCMV develop a persistent tolerant infection characterized by continuous viral production. Virus can be found in various body organs including lung, liver, kidney, and brain. In brain, neurons are the predominant CNS cells infected and the greatest number of persistently infected neurons are found in the cerebral cortex, hippocampus, other limbic structures and parts of the hypothalamus. Despite continuous infection throughout the animal's life, neurons show no structural injury or dropout. Mice from the DBA/2J strain were infected with LCMV (1000 plaque-forming units) within 18 h of birth and tested for behavioral function as adults. Plaque assays indicated persistent infection in virus-injected mice. Mice were tested for their ability to learn a Y-maze spatial discrimination to avoid the onset of a mild footshock (0.43 mA). The number of correct avoidance responses made during training was taken as a measure of acquisition performance. The virus-infected mice showed a deficit in acquisition of the Y-maze discrimination compared to that seen in vehicle-injected and noninjected controls. Following additional training to reach control levels of performance, the infected mice and the controls were injected with the cholinergic antagonist scopolamine. Scopolamine (2.0 mg/kg) disrupted the performance of the infected mice significantly more than control performance, suggesting that a cholinergic dysfunction accounted for some of the learning deficit. A separate group of virus-infected mice exhibited hypoactivity during the first exposure to a locomotor testing apparatus.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Nuclear Sm antigens in the sperm of different organisms.
Immunoblot analysis of sperm protein from several species revealed the presence of polypeptides recognised by anti-Sm sera obtained from patients with systemic lupus erythematosus. Immunoreactive polypeptides in human, bull, mouse and rat sperm were identified as protein B', B and D as compared with the Sm polypeptides of HeLa cells. In the sperm of rooster, the teleost fish Cyprinus carpio and the mussel Choromytilus chorus, the immunoreactive polypeptide profile was more complex. To ascertain the sperm origin of the Sm antigens, immunolocalisation with anti-Sm serum was carried out. The results demonstrated that in all the species studied staining was confined to the sperm nucleus, confirming that some polypeptides of the small nuclear ribonucleoprotein complex are present in the gamete.
Animals↗
Induction and habituation of immediate early gene expression in rat brain by acute and repeated restraint stress.
Acute exposure to stress leads to activation of the pituitary-adrenal axis (PA-axis) while repeated exposure to a homotypic stressor generally results in habituation of this response. Previous studies suggested that such habituation is largely due to changes in afferents of the PA-axis. To examine where within these afferents habituation occurs, we studied the effect of acute and repeated exposure to 2 hr restraint stress on expression of c-fos mRNA, as a marker of altered neuronal activity, in brain regions previously shown to influence the activity of the PA-axis. Acute restraint stress increased expression of c-fos mRNA in cortex, hippocampus, hypothalamus, septum, and brainstem. In contrast, the effect of restraint stress on c-fos expression in the aforementioned brain regions was much smaller in animals restrained once daily for 4 d, and nonexistent in animals restrained once daily for 9 d. A similar pattern of induction and habituation of jun-B, but not zif-268, c-jun, or jun-D mRNA expression, was observed in the cortex of animals exposed to acute versus repeated restraint stress. The habituation of c-fos responses was stressor specific: exposure of restraint-adapted animals to a novel (20 min swim) stress produced an increase in levels of c-fos mRNA in every examined brain region comparable to that seen in animals exposed to this stressor for the first time. Adrenalectomy did not alter the pattern of c-fos expression induced by acute and repeated restraint stress. Therefore, activation and habituation of these c-fos responses are independent of changes in circulating levels of corticosterone.
Adrenalectomy↗
[Status of portal perfusion in colorectal cancer. Swiss Study Group for Clinical Cancer Research].
533 patients with diagnosis of operable colorectal carcinoma were randomized to receive either a single course of portal infusion with Mitomycin-C (MMC) and 5-Fluorouracil (5-FU) starting immediately after operation, or no adjuvant treatment. Of these, 505 (94%) were evaluable. Over the median follow-up of 8 years, the adjuvant therapy reduced the risk of recurrence by 22% (Hazard ratio = 0.78%, 95% CI 0.61-0.99; P = 0.045). The relative reduction of relapse on death was similar in all subgroups (i.e. nodal status, localization). However, adjuvant portal chemotherapy proved to be most efficient in the subgroups of patients with tumor involvement of the regional lymph nodes (Dukes C) and of patients with colon cancer. Analysis of the pattern of relapse showed that most of the difference in overall and disease-free survival is to be attributed to a consistent reduction of all kinds of tumor recurrences (i.e. local relapses, liver metastases and/or other distant metastases) in the treated group, rather than to liver relapses alone. We conclude therefore, that part of significant benefit obtained for patients with operable colorectal carcinoma treated with a single course of adjuvant chemotherapy via the portal vein might be due to the additional systemic effects of the portal chemotherapy and further study of perioperative treatment with and without prolonged chemotherapy appears worthwhile.
Adenocarcinoma↗
Splice-site selection and decoding: are they related?
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Aminoglycosides↗
Lead cleavage sites in the core structure of group I intron-RNA.
Self-splicing of group I introns requires divalent metal ions to promote catalysis as well as for the correct folding of the RNA. Lead cleavage has been used to probe the intron RNA for divalent metal ion binding sites. In the conserved core of the intron, only two sites of Pb2+ cleavage have been detected, which are located close to the substrate binding sites in the junction J8/7 and at the bulged nucleotide in the P7 stem. Both lead cleavages can be inhibited by high concentrations of Mg2+ and Mn2+ ions, suggesting that they displace Pb2+ ions from the binding sites. The RNA is protected from lead cleavage by 2'-deoxyGTP, a competitive inhibitor of splicing. The two major lead induced cleavages are both located in the conserved core of the intron and at phosphates, which had independently been demonstrated to interact with magnesium ions and to be essential for splicing. Thus, we suggest that the conditions required for lead cleavage occur mainly at those sites, where divalent ions bind that are functionally involved in catalysis. We propose lead cleavage analysis of functional RNA to be a useful tool for mapping functional magnesium ion binding sites.
Base Sequence↗
RNA as a catalyst: natural and designed ribozymes.
RNA can catalyse chemical reactions through its ability to fold into complex three-dimensional structures and to specifically bind small molecules and divalent metal ions. The 2'-hydroxyl groups of the ribose moieties contribute to this exceptional reactivity of RNA, compared to DNA. RNA is not only able to catalyse phosphate ester transfer reactions in ribonucleic acids, but can also show amino-acyl esterase activity, and is probably able to promote peptide bond formation. Bearing its potential for functioning both as a genome and as a gene product, RNA is suitable for in vitro evolution experiments enabling the selection of molecules with new properties. The growing repertoire of RNA catalysed reactions will establish RNA as a primordial molecule in the evolution of life.
Base Sequence↗
U1 and U2 snRNA are localized in the sperm nucleus.
Analysis of epididymal rat sperm RNA strongly suggested the presence of U snRNAs, especially U1 and U2 snRNA. By Northern blot analysis with radiolabeled oligodeoxynucleotide probes, the presence of U1 and U2 snRNA in rat sperm was confirmed. To precisely localize these RNAs, in situ hybridization with antisense and sense oligo probes labeled with digoxigenin was carried out. The results indicate that U1 as well as U2 snRNA are confined to the sperm nucleus.
Animals↗
[Association of a giant Meckel's diverticulum and Crohn disease of the neighboring intestinal segments].
We report the case of a 51-year old patient presenting a giant Meckel's diverticulum associated with Crohn's disease in the proximal and the distal segments. The voluminous diverticulum had already been demonstrated by radiographies at the age of 28 years. Late symptoms were signs of partial obstruction and malabsorption.
Anastomosis, Surgical↗
Non-competitive inhibition of group I intron RNA self-splicing by aminoglycoside antibiotics.
Aminoglycoside antibiotics inhibit self-splicing of group I intron RNA in vitro at concentrations as low as 10(-6) M. The sites of interaction and the mechanism of inhibition have yet to be determined. A comparative study of inhibition by different 2-deoxystreptamine analogues reveals structural features of the aminoglycoside antibiotics required for their interaction and effect on group I introns. Complete antibiotic inhibition of the two steps of splicing was not reversed at high concentrations of guanosine, indicating a non-competitive inhibition. A mutant group I intron in which the conserved guanosine nucleotide of the G-binding site had been replaced by an adenosine, was sensitive to the antibiotics providing direct evidence that the antibiotics do not interact with the G-binding site in the same way as the guanine base. In addition kinetic analyses of the self-splicing process in the presence of antibiotic inhibitors supported a non-competitive mechanism of the mixed type for inhibition of the ribozyme.
Aminoglycosides↗
Choroidal neovascularization associated with choroidal hemangiomas.
Two patients with choroidal hemangiomas developed choroidal neovascularization. One patient with Sturge-Weber syndrome, a unilateral diffuse choroidal hemangioma, and a serous detachment of the macula was treated with yellow dye laser photocoagulation in a grid pattern to the tumor before glaucoma filtration surgery. Four years after successful laser treatment, the patient developed subfoveal choroidal neovascularization in an area of treatment. A second patient with a circumscribed choroidal hemangioma developed spontaneous subfoveal choroidal neovascularization 12 years after initial diagnosis of the hemangioma. The development of choroidal neovascularization associated with choroidal hemangiomas represents a potential cause of poor visual outcome in these patients.
Adolescent↗
Evolution of secondary metabolite production: potential roles for antibiotics as prebiotic effectors of catalytic RNA reactions.
It has been proposed that organic molecules related to known secondary metabolites have existed since the beginning of biochemical evolution and were present in primordial soups. Under primitive earth conditions certain of these molecules may have played roles as effectors in prebiotic reactions, especially those involving catalytic RNA (ribozymes). We demonstrate that a number of antibiotic-related secondary metabolites bind to group I introns and either inhibit splicing reactions or promote the formation of intron oligomers. This is consistent with the functional co-evolution of catalytic RNA and secondary metabolites as antibiotic inhibitors of translation, and supports the notion of an evolutionary relationship between group I introns and ribosomal RNA.
Anti-Bacterial Agents↗
Antibiotic inhibition of group I ribozyme function.
The discovery of catalytically active RNA has provided the basis for the evolutionary concept of an RNA world. It has been proposed that during evolution the functions of ancient catalytic RNA were modulated by low molecular weight effectors, related to antibiotics, present in the primordial soup. Antibiotics and RNA may have coevolved in the formation of the modern ribosome. Here we report that a set of aminoglycoside antibiotics, which are known to interact with the decoding region of the 16S ribosomal RNA of Escherichia coli, inhibit the second step of splicing of the T4 phage-derived td intron. Thus catalytic RNA seems to interact not only with a mononucleotide and an amino acid, but also with another class of biomolecules, the sugars. Splicing of other group I introns but not group II introns was inhibited. The similarity in affinity and specificity of these antibiotics for group I introns and rRNAs may result from recognition of evolutionarily conserved structures.
Animals↗
Streptomycin inhibits splicing of group I introns by competition with the guanosine substrate.
Streptomycin is an aminocyclitol glycoside antibiotic, which interferes with prokaryotic protein synthesis by interacting with the ribosomal RNA. We report here that streptomycin is also able to inhibit self splicing of the group I intron of the thymidylate synthase gene of phage T4. The inhibition is kinetically competitive with the substrate guanosine. Streptomycin and guanosine have in common a guanidino group, which has been shown to undergo hydrogen bonds with the ribozyme (Bass & Cech, Biochemistry, 25, 1986, 4473). The inhibitory effect of streptomycin extends to other group I introns, but does not affect group II introns. Mutating the bulged nucleotide in the conserved P7 secondary structure element of the td intron alters the affinity of the ribozyme for both guanosine and streptomycin. Myomycin, an antibiotic with similar effects on protein synthesis as streptomycin, is also able to inhibit splicing. In contrast, bluensomycin, which is structurally related to streptomycin, but contains only one guanidino group does not inhibit splicing. We discuss these findings in support of an evolutionary model that stresses the antiquity of antibiotics (J. Davies, Molecular Microbiology 4, 1990, 1227).
Aminoglycosides↗
Effects of mutations of the bulged nucleotide in the conserved P7 pairing element of the phage T4 td intron on ribozyme function.
The P7 element of group I introns contains a semiconserved "bulged" nucleotide, a C in group IA introns (nt 870 in the td intron) and an A in group IB introns [Cech, T.R. (1988) Gene 73, 259-271]. Variants U870, G870, and A870, isolated by a combination of in vitro and in vivo genetic strategies, indicate that C and A at position 870 are consistent with splicing whereas U and G are not. Although mutants G870 and U870 could be activated in vitro by increasing the Mg2+ concentration, their Km for GTP at pH 7 was 20-100-fold elevated, and they were unable to undergo site-specific hydrolysis. The dependence of the mutants on high guanosine concentrations could be substantially overcome by an increase in pH, suggesting that a tautomeric change, which makes U and G mimic C and A, is responsible for restoring function. In contrast to the striking Km effect, Vmax for the mutants differed by less than a factor of 2 from the wild type. Furthermore, streptomycin, an aminoglycoside antibiotic that competes with guanosine for its binding site, inhibited splicing of the U870 and G870 constructs at least as well as of the C870 and A870 variants, indicating that the guanosine-binding site of the mutants is proficient at interacting with a guanidino group. While our experiments argue against a hydrogen-bonding interaction between the C6-O of the cofactor and C4-NH2 of the bulged nucleotide, they are consistent with other models in which the C4-NH2 and/or N3 groups of the bulged C are involved in establishing an active ribozyme.
Base Sequence↗
Streptomycin and self-splicing.
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Binding, Competitive↗