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At least 1,153 records · Page 64Linked to original sources
PubMed1997

Incorporation of beta-globin untranslated regions into a Sindbis virus vector for augmentation of heterologous mRNA expression.

Polynucleotide immunization has been employed as a means of inducing immune responses through the introduction of antigen-encoding DNA. While immunization against specific tumor antigens may be achieved through this strategy, various candidate tumor antigens may not be approached via DNA-based vaccines as they represent transforming oncogenes. As an alternative approach, we have explored the utility of mRNA vectors for polynucleotide immunization. The transient expression achieved by mRNA may provide an efficient and safe system for stimulating immune responses to tumor-specific antigens. Our previous work demonstrated that a self-replicating RNA enhances the magnitude and duration of transgene expression for this application. Here we further modify the vector for optimal use in gene therapy through the incorporation of untranslated regions flanking the encoded transgene. The beta-globin 5' and 3' untranslated regions (UTRs) were inserted directly flanking the luciferase gene in both nonreplicative and replicative RNA constructs. In both cases, elevated and prolonged levels of luciferase expression were detected from the beta-globin UTR-flanked luciferase as compared to luciferase without these sequences. These modifications improve the ability of replicative RNA vectors to produce high, yet transient transgene expression for cancer immunotherapy strategies.

Animals↗
PubMed2000

Mechanistic studies of RNA modifying enzymes. RNA pseudouridine synthase and m5cytosine methyl transferase.

RNA has numerous post-transcriptional modifications, but relatively little is known about the enzymes that catalyze such modifications or about the functions of the modified residues. Our laboratory has been engaged in studies of the structure and function of enzymes that catalyze the conversion of Urd residues in RNA to pseudouridine (psi Synthase), and Cyd to 5-methylcytidine methyl transferase (RNA m5C Mtase). The presentation will summarize recent results from our laboratory.

Amino Acid Motifs↗
PubMed1983

Analysis of human rotavirus mixed electropherotypes.

Mixed human rotavirus electropherotypes were detected in stool samples from patients with acute gastroenteritis in Santiago, Chile. These electropherotypes accounted for 10% of 149 samples studied. The finding of extra RNA fragments with respect to the regular 11 genome segments suggests the possibility of simultaneous or sequential infection by more than one electropherotype in a single diarrhea event or occurrence of modification in the length of the RNA segments during an infection. These possibilities arose from gel electrophoretic analysis of unique and sequential samples of human rotavirus genome RNA.

Animals↗
PubMed1980

Implication of arginyl residues in mRNA binding to ribosomes.

Modification of Escherichia coli robosomes with phenylglyoxal and butanedione, protein reagents specific for arginyl residues, inactivates polypeptide polymerization, assayed as poly(U)-dependent polyphenylalanine synthesis, and the binding of poly(U). Inactivation is produced by modification of the 30-S subunit. Both the RNA and the protein moieties of 30-S subunits are modified by phenylglyoxal, and modification of either of them is accompanied by inactivation of polypeptide synthesis. Modification of only the split proteins released from 30-S subunits by prolonged dialysis against a low-ionic-strength buffer, which contain mainly protein S1, produces inhibition of poly(U) binding and inactivation of polypeptide synthesis. Amino acid analysis of the modified split proteins showed a significant modifications of arginyl residues. These results indicate that the arginyl residues of a few 30-S proteins might be important in the interaction between mRNA and the 30-S subunit, which agrees with the general role assigned to the arginyl residues of proteins as the positively charged recognition site for anionic ligands.

Arginine↗
PubMed2002

Aminoglycosides modified by resistance enzymes display diminished binding to the bacterial ribosomal aminoacyl-tRNA site.

Understanding the basic principles that govern RNA binding by aminoglycosides is important for the design of new generations of antibiotics that do not suffer from the known mechanisms of drug resistance. With this goal in mind, we examined the binding of kanamycin A and four derivatives (the products of enzymic turnovers of kanamycin A by aminoglycoside-modifying enzymes) to a 27 nucleotide RNA representing the bacterial ribosomal A site. Modification of kanamycin A functional groups that have been directly implicated in the maintenance of specific interactions with RNA led to a decrease in affinity for the target RNA. Overall, the products of reactions catalyzed by aminoglycoside resistance enzymes exhibit diminished binding to the A site of bacterial 16S rRNA, which correlates well with a loss of antibacterial ability in resistant organisms that harbor these enzymes.

Anti-Bacterial Agents↗
PubMed1995

Accurate modification of the trypanosome spliced leader cap structure in a homologous cell-free system.

During RNA maturation in trypanosomatid protozoa, trans-splicing transfers the spliced leader (SL) sequence and its cap from the SL RNA to the 5' end of all mRNAs. In Trypanosoma brucei and Crithidia fasciculata the SL RNA has an unusual cap structure with four methylated nucleotides following the 7-methylguanosine residue (cap 4). Since modification of the 5' end of the SL RNA is a pre-requisite for trans-splicing activity in T. brucei, we have begun to characterize the enzyme(s) involved in this process. Here we report the development of a T. brucei cell-free system for modification of the cap of the SL RNA. Analysis of the nucleotide composition of the in vitro generated cap structure by two-dimensional thin layer chromatography established that the in vitro reaction is accurate. Cap 4 formation requires the SL RNA to be in a ribonucleoprotein particle and can be inhibited by annealing a complementary 2'-O-methyl RNA oligonucleotide to nucleotides 7-18 of the SL RNA. Methylation of the 5' end of the SL RNA is also required for trans-splicing in T. cruzi and Leishmania amazonensis and cell-free extracts from C. fasciculata and L. amazonensis are capable of modifying the cap structure on the T. brucei SL ribonucleoprotein particle.

Adenosine Triphosphate↗
PubMed1996

Nuclear transplantation of ectodermal cells in pig oocytes: ultrastructure and radiography.

This study investigated the changes in nucleolar fine structure and the synthesis of both heterogeneous nuclear RNA (hnRNA) and ribosomal RNA (rRNA) in pig reconstructed embryos obtained by electrofusion of a single pig ectodermal cell to an enucleated metaphase-II oocyte. The nucleolar morphological changes and the pattern of transcription were examined in the ectodermal cells before fusion and in the nuclear transfer reconstructed embryos 16-18 hr after fusion. Before fusion the ectodermal cells exhibited a reticulated nucleolus with active RNA synthesis. In the reconstructed embryos, modifications of the nucleolar structure were observed, as assessed by the presence of either round-shaped, compact, dense nucleolar precursor bodies, or reticulated nucleoli. However, in both cases there was no RNA synthesis. Blebbing activity of the nuclear envelope was not observed. These results indicate that the nucleus of pig ectodermal cells exhibited either a complete or an incomplete remodelling when transferred to an enucleated metaphase-II oocyte, with no detectable RNA synthesis. Cell-cycle synchronization of ectodermal cell donor nuclei may play an important role in nuclear reprogramming after fusion.

Animals↗
PubMed1993

Hydrogen-bonding contacts in the major groove are required for human immunodeficiency virus type-1 tat protein recognition of TAR RNA.

The binding site for tat on TAR RNA was analysed by preparing a series of model RNA substrates carrying site-specific functional group modifications. The test RNAs were prepared by annealing two short synthetic oligoribonucleotides to form a duplex structure with a U-rich bulge and flanking sequences identical to TAR RNA. Tat binds these duplex RNAs with approximately half the affinity for wild-type TAR RNA. Substitution at positions U23 or U25 by the base analogue, O4-methyl-dT, which is deficient in its ability to hydrogen-bond at the N3 position reduces tat affinity more than 20-fold. Modifications to purines in the stem of TAR RNA that affect hydrogen-bonding ability in either the major or the minor groove of duplex RNA were also tested. Removal of the nitrogen atom at either the N7 position of G26 or at the N7 position of A27 reduces tat affinity 10- to 20-fold. By contrast removal of the exocyclic amino group in the minor groove at position G26, by substitution with inosine, does not affect tat binding significantly. A single methylphosphonate substitution at the phosphate bond between A22 and U23 also leads to a significant loss of tat binding ability, whereas all other methylphosphonate substitutions in the U-rich bulge are not harmful to tat binding. We conclude that tat forms multiple specific hydrogen bonds to a series of dispersed sites displayed in the major groove of the TAR RNA molecule. These include the N3-H of U23, the N7 of G26, the N7 of A26 and the phosphate between A22 and U23.

Base Sequence↗
PubMed1999

Molecular modeling of the three-dimensional architecture of the RNA component of yeast RNase MRP.

RNase mitochondrial RNA processing (MRP) is a ribonucleoprotein endoribonuclease that is involved in RNA processing events in both the nucleus and the mitochondria. The MRP RNA is both structurally and evolutionarily related to RNase P, the ribonucleoprotein endoribonuclease that processes the 5'-end of tRNAs. Previous analysis of the RNase MRP RNA by phylogenetic analysis and chemical modification has revealed strikingly conserved secondary structural elements in all characterized RNase MRP RNAs. Utilizing successive constraint modeling and energy minimization I derived a three-dimensional model of the yeast RNase MRP RNA. The final model predicts several notable features. First, the enzyme appears to contain two separate structural domains, one that is highly conserved among all MRP and P RNAs and a second that is only conserved in MRP RNAs. Second, nearly all of the highly conserved nucleotides cluster in the first domain around a long-range interaction (LRI-I). This LRI-I is characterized by a ubiquitous uridine base, which points into a cleft between these two structural domains generating a potential active site for RNA cleavage. Third, helices III and IV (the yeast equivalent of the To-binding site) model as a long extended helix. This region is believed to be the binding site of shared proteins between RNase P and RNase MRP and would provide a necessary platform for binding these seven proteins. Indeed, several residues conserved between the yeast MRP and P RNAs cluster in the central region of these helixes. Lastly, characterized mutations in the MRP RNA localize in the model based on their severity. Those mutations with little or no effect on the activity of the enzyme localize to the periphery of the model, while the most severe mutations localize to the central portion of the molecule where they would be predicted to cause large structural defects. Press.

Bacillus subtilis↗
PubMed1956

The action of ultraviolet radiation on yeast catalase.

The effect of prolonged UV irradiation (mostly 2537 A) on the catalase activity of an aqueous yeast suspension was divisible into 4 periods. First, the period during which the cells lost their ability to form colonies, but during which no change in catalase activity was noted. Second, the period during which a considerable rise in catalase activity (Euler effect) occurred. The Euler effect was accompanied by enzyme alteration as shown by the simultaneous decrease in the activation energy of the enzyme-substrate system. However, during the initial phase of this period, as the catalase activity of the suspension began to increase, the activation energy rose to a transient level higher even than that characterizing the unaltered enzyme. Heat accelerated the rate of alteration when applied either during or after the irradiation; the activation energy for the over-all alteration reaction was 24 kcal., a value close to that recorded previously for alteration induced by chemical agents. Nevertheless, the rate-limiting step appeared to be different in the two cases. A model of these events was presented in which the primary photochemical action was on the site at which catalase is located within the cell. Third, a rather long period during which irradiation led to no diminution in the catalase activity of the maximally active suspension. This protection effect was duplicated in intro by a model crystalline catalase-KNA system, or by adding either ribonuclease digestion products of RNA or adenine to a catalase solution prior to irradiation. Evidence was adduced that the protection effect was not a simple screening, but involved some sort of interaction between the enzyme and the nitrogenous components of RNA, an interaction which must likewise occur within the cell. Alteration induced by CHCl(3) did not eliminate the protection effect, but that by butanol did. The onset of photoinactivation was due to modification of protein structure, not of RNA. Fourth, the period of photoinactivation of the intracellular enzyme, which was quite similar to that of the crystalline enzyme in vitro.

Catalase↗
PubMed2003

Silencing of Sm proteins in Trypanosoma brucei by RNA interference captured a novel cytoplasmic intermediate in spliced leader RNA biogenesis.

In Trypanosoma brucei the small nuclear (sn) RNAs U1, U2, U4, and U5, as well as the spliced leader (SL) RNA, bind the seven Sm canonical proteins carrying the consensus Sm motif. To determine the function of these proteins in snRNA and SL RNA biogenesis, two of the Sm core proteins, SmE and SmD1, were silenced by RNAi. Surprisingly, whereas the level of all snRNAs, including U1, U2, U4, and U5 was reduced during silencing, the level of SL RNA was dramatically elevated, but the levels of U6 and spliced leader-associated RNA (SLA1) remained unchanged. The SL RNA that had accumulated in silenced cells lacked modification at the cap4 nucleotide but harbored modifications at the cap1 and cap2 nucleotides and carried the characteristic psi. This SL RNA possessed a longer tail and had accumulated in the cytoplasm in 10 and 50 S particles that were found by in situ hybridization to be present in "speckles." We propose a model for SL RNA biogenesis involving a cytoplasmic phase and suggest that the trypanosome-specific "cap4" nucleotides function as a signal for export and import of SL RNA out and into the nucleus. The SL RNA biogenesis pathway differs from that of U sn ribonucleoproteins (RNPs) in that it is the only RNA that binds Sm proteins that were stabilized under Sm depletion in a novel RNP, which we termed SL RNP-C.

Active Transport, Cell Nucleus↗
PubMed1984

Primary and secondary structure of dinoflagellate U5 small nuclear RNA.

U5 RNA is one of the six capped small nuclear RNAs present in most eukaryotic cells. Like U1, U2, U4 and U6 RNAs, U5 RNA is associated with hnRNP particles and is thus probably involved in some, as yet undefined, aspects of pre-messenger RNA processing. In this study, the complete nucleotide sequence of U5 RNA of a dinoflagellate, Crypthecodinium cohnii was determined. The analysis of this dinoflagellate U5 RNA sequence showed that a) the sequence homology between human, rat and chicken U5 RNA sequences and dinoflagellate U5 RNA sequence is 64%; b) the extent and the position of post-transcriptional modifications are similar to those found in U5 RNA of higher eukaryotes; c) although the dinoflagellate U5 RNA is shorter in length (108 nucleotides long vs 117 long in human, rat and chicken cells), the RNA fits well into the same secondary structure proposed for U5 RNA of higher eukaryotes (Krol et al. (1981) Nucl. Acids Res. 9, 769); and d) the AUn nucleotide sequence protected by the Sm-antigen and the tight secondary structure found near the 3'-end of other U-RNAs was also found in dinoflagellate U5 RNA. The high order of homology observed between dinoflagellate U5 RNA and U5 RNA of higher eukaryotes indicates that dinoflagellates are more closely related to metazoans than to early eukaryotes.

Animals↗
PubMed2004

Cyclosporin induces renal proto-oncogene RNA message and increased transforming growth factor-beta prior to renal fibrosis: Modification by calcium channel blockade in the salt replete rat.

BACKGROUND: Chronic cyclosporin (CsA) administration has been shown to result in the replacement of epithelial cells in the kidney with fibrous tissue. These changes are kidney-specific, as they do not occur in any other organ. RESULTS: Cyclosporin exposure increases c-fos and c-jun mRNA in the rat kidney but not in the liver. Furthermore, chronic CsA exposure causes a further increase in c-fos and c-jun mRNA and increases the renal expression of transforming growth factor-beta (TGF-beta) mRNA. These changes precede the development of fibrosis. The combined insult of ischaemia and CsA resulted in synergistic increases in c-fos, suggesting that CsA recruited a pathway for c-fos activation different from ischaemia. The calcium channel blocker, verapamil, blocked CsA-induced expression of c-fos and c-jun mRNA, and reduced the amount of TGF-beta expression. CONCLUSION: These data are consistent with the notion that CsA induces protooncogenes, which may be, at least partially, responsible for long-term CsA nephrotoxicity.

Animals↗
PubMed1987

Expression of bacterial chloramphenicol acetyltransferase gene in tobacco plants mediated by TMV-RNA.

We have constructed three tobacco mosaic virus (TMV) cDNA derivatives by modification of the full-length cDNA clone from which infectious TMV-RNA can be transcribed in vitro. A coatless TMV construct lacks most of the coat protein gene and chimeric TMV constructs retain the bacterial chloramphenicol acetyltransferase (CAT) gene in place of the coat protein gene. When in vitro transcripts from these cDNA derivatives were inoculated on the local lesion tobacco plants, TMV-specific lesions were produced. In the case of the TMV-CAT chimeras, however, the lesions were small compared to those of wild-type TMV and those produced by transcript derived from the coatless construct. Northern blot analysis of RNA extracted from the inoculated leaves of the systemic host plants revealed replication of the derivative genomic RNAs and production of their own subgenomic RNAs corresponding to the coat protein mRNA. The TMV-CAT chimeras produced biologically active CAT in the inoculated leaves of the systemic host. CAT activity increased at least until 2 weeks post-inoculation and was approximately 0.1 units/mg of tissue at 10 days post-inoculation. Thus, TMV-RNA may be utilized as a new plant expression vector.

Blotting, Northern↗
PubMed1996

Spliced leader RNA of trypanosomes: in vivo mutational analysis reveals extensive and distinct requirements for trans splicing and cap4 formation.

In trypanosomes mRNAs are generated through trans splicing. The spliced leader (SL) RNA, which donates the 5'-terminal mini-exon to each of the protein coding exons, plays a central role in the trans splicing process. We have established in vivo assays to study in detail trans splicing, cap4 modification, and RNP assembly of the SL RNA in the trypanosomatid species Leptomonas seymouri. First, we found that extensive sequences within the mini-exon are required for SL RNA function in vivo, although a conserved length of 39 nt is not essential. In contrast, the intron sequence appears to be surprisingly tolerant to mutation; only the stem-loop II structure is indispensable. The asymmetry of the sequence requirements in the stem I region suggests that this domain may exist in different functional conformations. Second, distinct mini-exon sequences outside the modification site are important for efficient cap4 formation. Third, all SL RNA mutations tested allowed core RNP assembly, suggesting flexible requirements for core protein binding. In sum, the results of our mutational analysis provide evidence for a discrete domain structure of the SL RNA and help to explain the strong phylogenetic conservation of the mini-exon sequence and of the overall SL RNA secondary structure; they also suggest that there may be certain differences between trans splicing in nematodes and trypanosomes. This approach provides a basis for studying RNA-RNA interactions in the trans spliceosome.

Animals↗
PubMed2001

The snoRNA domain of vertebrate telomerase RNA functions to localize the RNA within the nucleus.

Telomerase RNA is an essential component of the ribonucleoprotein enzyme involved in telomere length maintenance, a process implicated in cellular senescence and cancer. Vertebrate telomerase RNAs contain a box H/ACA snoRNA motif that is not required for telomerase activity in vitro but is essential in vivo. Using the Xenopus oocyte system, we have found that the box H/ACA motif functions in the subcellular localization of telomerase RNA. We have characterized the transport and biogenesis of telomerase RNA by injecting labeled wild-type and variant RNAs into Xenopus oocytes and assaying nucleocytoplasmic distribution, intranuclear localization, modification, and protein binding. Although yeast telomerase RNA shares characteristics of spliceosomal snRNAs, we show that human telomerase RNA is not associated with Sm proteins or efficiently imported into the nucleus. In contrast, the transport properties of vertebrate telomerase RNA resemble those of snoRNAs; telomerase RNA is retained in the nucleus and targeted to nucleoli. Furthermore, both nuclear retention and nucleolar localization depend on the box H/ACA motif. Our findings suggest that the H/ACA motif confers functional localization of vertebrate telomerase RNAs to the nucleus, the compartment where telomeres are synthesized. We have also found that telomerase RNA localizes to Cajal bodies, intranuclear structures where it is thought that assembly of various cellular RNPs takes place. Our results identify the Cajal body as a potential site of telomerase RNP biogenesis.

Active Transport, Cell Nucleus↗
PubMed2005

Histone modifications as key regulators of transcription.

Covalent modifications of the amino-termini of the core histones in nucleosomes have been shown to be one of the key regulatory mechanisms in transcription regulation. Recently, new roles for histone modifications have been uncovered for the efficient functioning of RNA Pol II. Besides acetylation, which is the most characterized to date these modifications comprise phosphorylation, methylation, and ubiquitination. This review gives comprehensive view of all the major histone modifications and their effect on transcriptional regulation, in Saccharomyces cerevisiae.

Acetylation↗
PubMed2003

Determination of the catalytic parameters of the N-terminal half of Escherichia coli ribonuclease E and the identification of critical functional groups in RNA substrates.

Ribonuclease E is required for the rapid decay and correct processing of RNA in Escherichia coli. A detailed understanding of the hydrolysis of RNA by this and related enzymes will require the integration of structural and molecular data with quantitative measurements of RNA hydrolysis. Therefore, an assay for RNaseE that can be set up to have relatively high throughput while being sensitive and quantitative will be advantageous. Here we describe such an assay, which is based on the automated high pressure liquid chromatography analysis of fluorescently labeled RNA samples. We have used this assay to optimize reaction conditions, to determine for the first time the catalytic parameters for a polypeptide of RNaseE, and to investigate the RNaseE-catalyzed reaction through the modification of functional groups within an RNA substrate. We find that catalysis is dependent on both protonated and unprotonated functional groups and that the recognition of a guanosine sequence determinant that is upstream of the scissile bond appears to consist of interactions with the exocyclic 2-amino group, the 7N of the nucleobase and the imino proton or 6-keto group. Additionally, we find that a ribose-like sugar conformation is preferred in the 5'-nucleotide of the scissile phosphodiester bond and that a 2'-hydroxyl group proton is not essential. Steric bulk at the 2' position in the 5'-nucleotide appears to be inhibitory to the reaction. Combined, these observations establish a foundation for the functional interpretation of a three-dimensional structure of the catalytic domain of RNaseE when solved.

Base Sequence↗