Biomedical subjects

S Rits

Publications and source records attributed to S Rits.

13 recordsLinked to original sources

PubMed1997

Hsp70 prevents activation of stress kinases. A novel pathway of cellular thermotolerance.

Harmful conditions including heat shock, oxidative stress, UV, and so forth cause programmed cell death, whose triggering requires activation of the Jun N-terminal kinase, JNK. High levels of Hsp72, a heat-inducible member of Hsp70 family, protect cells against a variety of stresses by a mechanism that is unclear at present. Here we report that elevated levels of Hsp72 inhibit a signal transduction pathway leading to programmed cell death by preventing stress-induced activation of JNK. Stress-induced activation of another stress-kinase, p38 (HOG1), is also blocked when the level of Hsp72 is increased. Similarly, addition of a purified recombinant Hsp72 to a crude cell lysate reduced p38 kinase activation, while depletion of the whole family of Hsp70 proteins with a monoclonal antibody enhanced such activation. In addition, we have found that accumulation of abnormal proteins in cells upon incubation with amino acid analogs causes activation of JNK and p38 kinases, which can be prevented by overproduction of Hsp72. Taken together, these data suggest that, in regulation of JNK and p38 kinases, Hsp70 serves as a "sensor" of the build-up of abnormal proteins after heat shock and other stresses. The inhibitory effect of an increased level of Hsp70 on JNK appears to be a major contributor to acquired thermotolerance in mammalian cells.

Apoptosis↗

PubMed1996

Antisense globin RNA in mouse erythroid tissues: structure, origin, and possible function.

The aim of the experiments described in this paper was to test for the presence of antisense globin RNA in mouse erythroid tissues and, if found, to characterize these molecules. The present study made use of a multistep procedure in which a molecular tag is attached to cellular RNA by ligation with a defined ribooligonucleotide. The act of ligation preserves the termini of RNA molecules, which become the junctions between cellular RNAs and the ligated ribooligonucleotide. It also unambiguously preserves the identity of cellular RNA as a sense or antisense molecule through all subsequent manipulations. Using this approach, we identified and characterized antisense beta-globin RNA in erythroid spleen cells and reticulocytes from anemic mice. We show in this paper that the antisense globin RNA is fully complementary to spliced globin mRNA, indicative of the template/transcript relationship. It terminates at the 5' end with a uridylate stretch, reflecting the presence of poly(A) at the 3' end of the sense globin mRNA. With respect to the structure of their 3' termini, antisense globin RNA can be divided into three categories: full-size molecules corresponding precisely to globin mRNA, truncated molecules lacking predominantly 14 3'-terminal nucleotides, and extended antisense RNA containing 17 additional 3'-terminal nucleotides. The full-size antisense globin RNA contains two 14-nt-long complementary sequences within its 3'-terminal segment corresponding to the 5'-untranslated region of globin mRNA. This, together with the nature of the predominant truncation, suggests a mechanism by which antisense RNA might give rise to new sense-strand globin mRNA.

Anemia↗

PubMed1995

Transcription of the 5'-terminal cap nucleotide by RNA-dependent DNA polymerase: possible involvement in retroviral reverse transcription.

The possible transcription of the 5'-terminal cap nucleotide of mRNA by RNA-dependent DNA polymerase was examined by a single-step assay, based on the generation of a hairpin structure during reverse transcription of globin mRNA. Using this approach, we demonstrated that the 5'-terminal cap nucleotide of mRNA is indeed transcribed by RNA-dependent DNA polymerase into a 3'-terminal residue of cDNA and were able to measure the extent of such transcription. The observed transcription of the cap nucleotide raises a number of questions that may be addressed using the relatively simple single-step assay employed in the present study. Cap nucleotide transcription by RNA-dependent DNA polymerase may have important implications for our understanding of the mechanism of action of reverse transcriptases. It may represent a selection mechanism for only partial transcription of the 5' repeat element of viral RNA genome, thus generating a RNA fragment that may play a role in priming of second (plus) DNA strand during retroviral reverse transcription. Moreover, the demonstrated ability of complementary nucleotides to form hydrogen bonds, even when in a parallel orientation, may have interesting and important consequences.

Animals↗

PubMed1994

Evolutionarily conserved elements in the 5' untranslated region of beta globin mRNA mediate site-specific priming of a unique hairpin structure during cDNA synthesis.

Generation of double-stranded cDNA during reverse transcription of a variety of mRNA molecules is well known to involve the formation of covalently linked antisense and sense strands in a hairpin configuration. In the present study we have examined the sequence of molecular events which occurs during cDNA synthesis from mouse beta globin mRNA, in particular the self-priming event that initiates synthesis of sense-strand DNA. Upon completion of reverse transcription of globin mRNA and the removal of RNA template by RNase H activity associated with reverse transcriptase, the 3' end of cDNA snaps back to form a stable double-stranded structure, which is extended by reverse transcriptase to generate the sense DNA strand. Surprisingly, the fourteen 3' terminal nucleotides of the beta globin antisense DNA strand (cDNA) have strong complementarity with an internal segment of the same molecule corresponding to a portion of the 5'-untranslated region of the mRNA located just upstream of the translation start site. Efficient second strand cDNA synthesis appears to require the occurrence within the cDNA molecule of these two complementary elements, one of which must be 3'-terminal. A second surprising feature is that the strong complementarity between the terminal and the internal portions of the molecule exists in the antisense DNA and not in the sense mRNA strand. This is because A:C mismatches on the sense strand correspond to relatively stable T:G base pairs on the antisense strand. Such an extended region of complementarity within the segment of cDNA corresponding to the short 5' untranslated region of beta globin mRNA is unlikely to occur purely by chance, suggesting some underlying function. In this regard it is of interest that cDNAs of adult beta globin mRNAs from other mammalian species show a very similar arrangement of complementary elements, and that complementarity is heavily conserved, even when there are substitutions in nucleotide sequence.

Animals↗

PubMed1994

Ligation-mediated amplification of RNA from murine erythroid cells reveals a novel class of beta globin mRNA with an extended 5'-untranslated region.

Ligation-mediated RNA amplification was developed as a tool for analysis and determination of the termini of RNA molecules [Volloch et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10671-10675]. In this approach, T4 RNA ligase is used to join cellular RNA with a defined ribo-oligonucleotide. Although several additional enzymatic steps are involved in this type of analysis, the reliability of the entire procedure is determined by the initial ligation step, which marks and preserves the termini of cellular RNA molecules. We applied this approach to the analysis of the 5' terminus of beta globin mRNA in various murine erythroid cells. As expected, we detected RNA molecules with 5' ends terminating at the regular cap site as well as globin RNA molecules truncated at the 5' end. Unexpectedly, we also detected a class of beta globin mRNA which is identical to regular beta globin mRNA in every respect but contains 17, 29, or 31 additional nucleotides 5' to the regular cap site. These extensions correspond precisely to the genomic segments just upstream of the regular cap site and are probably generated by initiation of transcription of the globin gene upstream from the regular cap site. It is likely that the extended globin RNA is transcribed not from the TATA promoter, which regulates the transcription of regular murine globin mRNA, but from the GATA regulatory element located 30 nucleotides upstream of the 31-nucleotide extension, in a position identical to that of the active GATA promoter of the TATA-less chicken beta globin gene. The evolutionary conservation of this relationship suggests the importance of the GATA promoter element of the mouse beta globin gene and its possible involvement in developmental regulation of expression of this gene.

Animals↗

PubMed1991

Identification of negative-strand complements to cytochrome oxidase subunit III RNA in Trypanosoma brucei.

A substantial amount of cytochrome oxidase subunit III (COIII) mRNA continues to be synthesized de novo in Trypanosoma brucei in the presence of actinomycin D, presumably by a DNA-independent transcription process. We describe the identification of negative-strand COIII RNA molecules, characterization of their termini, and the detection of RNA-dependent RNA polymerase activity. Three lines of evidence for the existence of negative-strand COIII RNA are presented: (i) hybridization with oligonucleotide probes with the same polarity as mRNA after preliminary enrichment for putative negative-strand RNA by affinity purification; (ii) cloning and sequencing of negative-strand complements for the unedited, edited, and partially edited COIII RNA; and (iii) exact correspondence of the terminal sequences of the putative negative-strand RNA molecules to the ends of COIII RNA. The presence of negative-strand complements of COIII RNA is consistent with the notion that a significant amount of mRNA in T. brucei is synthesized by an RNA-dependent RNA polymerase with negative-strand RNA as an intermediate template.

Animals↗

PubMed1991

Inhibition of pre-mRNA splicing by antisense RNA in vitro: effect of RNA containing sequences complementary to exons.

The objective of the experiments described in this paper was to determine the feasibility of inhibition of pre-mRNA splicing by antisense RNA in vitro. Three different types of antisense RNA were utilized: antisense RNA complementary to the spliced RNA molecule; antisense RNA complementary to the unprocessed mRNA precursor molecule; and antisense RNA complementary to the 5' and 3' splice junctions. Whereas antisense RNA complementary to mRNA had little effect on splicing, antisense RNAs complementary to mRNA precursor or to splice junctions strongly inhibited splicing of pre-mRNA molecule. The results obtained indicate that the inhibitory effect is most likely due to hybrid formation between pre-mRNA and antisense RNA molecules and that antisense RNA complementary to the exon portion but not to the intron portion of splice junction exhibit an inhibitory effect. This inhibition can be overcome by bringing together 5' and 3' splice junctions via hybrid formation with antisense RNA complementary to the spliced RNA molecule.

Animals↗

PubMed1991

Inhibition of pre-mRNA splicing by antisense RNA in vitro: effect of RNA containing sequences complementary to introns.

The objective of the experiments described in this paper was to test the potential of antisense RNAs complementary to the internal portion of an intron to inhibit the splicing process and to determine the mechanism of such inhibition. The results obtained indicate that RNA fragments complementary to the internal portion of an intron can effectively inhibit the splicing of pre-mRNA. Inhibition was observed only with antisense RNA complementary to pre-mRNA suggesting that the inhibitory effect was due to the formation of a hybrid with the corresponding portion of the pre-mRNA's intron. The observed inhibition was not due to interference with possible intron elements essential for the splicing process, for the deletion of the sequences complementary to inhibitory antisense RNA from the corresponding pre-mRNA molecule did not affect the efficiency of a splicing reaction, and the addition of antisense RNA to pre-mRNA mutants carrying such deletions did not result in any inhibition. Our results indicate that the observed inhibition is a function of the length of the antisense RNA expressed as a fraction of an intron with which it interacts when antisense RNA is modified by incorporation of a "hinge" element, it loses its inhibitory potential suggesting that the inhibitory effect is probably due to limitation of conformational flexibility of an intron.

Animals↗

PubMed1990

Uncoupling of the synthesis of edited and unedited COIII RNA in Trypanosoma brucei.

RNA editing, a novel and unexpected type of information processing, was first demonstrated in the kinetoplasts of certain protozoans. It is a remarkable phenomenon: certain species of messenger RNA have nucleotide sequences that differ greatly from the sequences of the genes from which they are presumably transcribed. The differences are usually due to addition of uridylate residues, but occasionally also to their deletion. The most spectacular case of editing known occurs in the mRNA of the mitochondrial gene for subunit III of cytochrome oxidase (COIII) in Trypanosoma brucei. This mRNA is twice the length of its gene owing to the addition of several hundred uridylate residues and a few deletions, spread over the entire length of the RNA molecule. Whereas unedited RNA molecules, the nucleotide sequences of which correspond to the genomic sequence, have been isolated, no DNA template corresponding to the edited RNA sequence has been detected in either the mitochondrial or nuclear genome. It was suggested, therefore, that unedited mRNAs are transcribed from mitochondrial DNA and then edited post-transcriptionally by endonucleolytic cleavage of the primary transcript at specific sites, followed by insertion or deletion of uridylate residues and religation. We have now examined the general nature of the RNA editing process to determine whether it involves the insertion of nucleotide residues into pre-existing molecules or the continuous de novo synthesis of edited mRNA. The results of our experiments rule out an insertion mechanism and strongly indicate that edited mRNA is synthesized as a unit.

Animals↗

PubMed1987

Messenger RNA changes during differentiation of murine erythroleukemia cells.

During differentiation of murine erythroleukemia cells, the levels of certain mRNA were observed to change. To characterize the various patterns of changes that occur during differentiation, cDNA libraries made from RNA isolated from uninduced and differentiating cells were screened with labeled cDNA or RNA labeled in vivo for different periods of time. cDNA clones that corresponded to individual mRNAs whose level remained constant, increased, or decreased during differentiation were identified. These clones were used to analyze Northern blots containing RNA from uninduced and differentiated cells. A number of characteristic changes in individual mRNAs in differentiating murine erythroleukemia cells could be identified, such as no change, increase in concentration, increase in concentration and slight change in size, decrease in concentration, decrease in concentration and change in size, appearance of new band(s) of entirely different size, and change in relative concentrations of two related mRNAs. Measurements of rates of mRNA synthesis and degradation suggest that both parameters change during differentiation and that these changes are instrumental in establishing cellular concentration of specific mRNAs. It seems that the changes in mRNA stability observed in differentiating murine erythroleukemia cells may be associated with changes in the primary structure of the transcribed portion of mRNA. The observation that specific mRNA synthesized before and after induction may have very different stabilities at the same point in differentiation supports this hypothesis.

Animals↗

PubMed1987

Synthesis of globin RNA in enucleated differentiating murine erythroleukemia cells.

In an earlier report (Volloch, V., 1986, Proc. Natl. Acad. Sci. USA., 83:1208-1212) we had presented evidence for the occurrence of the cytoplasmic synthesis of globin mRNA and of RNA complementary to globin mRNA which differed from DNA-dependent transcription by its insensitivity to actinomycin D. In this paper, we describe the use of enucleated differentiating mouse erythroleukemia cells to demonstrate directly the occurrence of cytoplasmic synthesis of both positive- and negative-strand globin RNA. For this purpose, we developed an enucleation procedure which yielded pure cytoplasts from differentiated mouse erythroleukemia cells in the absence of cytochalasin B and selectively permeabilized the cytoplasts to small molecules by treatment with dextran sulfate and saponin. The permeabilized cytoplasts incorporated [3H]dUTP into positive- and negative-strand globin RNA and experiments with mercurated nucleotide substrate suggested that this process involved de novo RNA synthesis rather than limited terminal nucleotide addition. Globin RNA synthesis required Mg++, was inhibited by Mn++, and was unaffected by the addition of Zn++. Studies of its response to inhibitors of DNA-dependent RNA synthesis showed that it differed from that process in its insensitivity to actinomycin D and alpha-amanitin, but that like many other macromolecular biosynthetic reactions it was inhibited by rifamycin AF/ABDP and aurintricarboxylic acid. These observations provide additional evidence for the occurrence of cytoplasmic RNA-dependent RNA synthesis in differentiated cells and show permeabilized enucleated cells to be a useful experimental system for further studies of the characteristics of that process.

Animals↗

PubMed1979

Pyrophosphate-condensing activity linked to nucleic acid synthesis.

In some preparations of DNA dependent RNA polymerase a new enzymatic activity has been found which catalyzes the condensation of two pyrophosphate molecules, liberated in the process of RNA synthesis, to one molecule of orthophosphate and one molecule of Mg (or Mn) - chelate complex with trimetaphosphate. This activity can also cooperate with DNA-polymerase, on condition that both enzymes originate from the same cells. These results point to two general conclusions. First, energy is conserved in the overall process of nucleic acid synthesis and turnover, so that the process does not require an energy influx from the cell's general resources. Second, the synthesis of nucleic acids is catalyzed by a complex enzyme system which contains at least two separate enzymes, one responsible for nucleic acid polymerization and the other for energy conservation via pyrophosphate condensation.

Chromatography, Thin Layer↗

PubMed1979

A possible mechanism responsible for the correction of transcription errors.

Nucleoside triphosphate phosphohydrolase (NTPase) activity was found in a preparation of E. Coli RNA polymerase. This enzymatic activity is capable of hydrolysing all four ribonucleoside triphosphates to the nucleoside diphosphates. However, during in vitro RNA synthesis directed by poly(dC) or poly(dT), only the non-complementary nucleoside triphosphate of the same heterocyclic class was hydrolysed. No incorporation of the non-complementary precursor into RNA could be detected in these experiments. When another RNA polymerase preparation, devoid of NTPase activity, was employed, there was no hydrolysis of any nucleoside triphosphate and significant incorporation of non-complemtary precursor into RNA was observed. These observations lead us to the conclusion that NTPase, acting in conjunction with RNA polymerase, has the function of correcting errors in transcription.

DNA-Directed RNA Polymerases↗