Oligonucleotide mapping of non-radioactive virus and messenger RNAs.

A modification of the known method for obtaining radioactive fingerprints from non-radioactive nucleic acids by labelling a digest with 5'-hydroxyl polynucleotide kinase and [gamma-32P]-ATP has been applied to RNase T1 digests from various high molecular weight virus RNAs and to ovalbumin mRNA. Fractionation of the resultant [32P]-labelled T1 RNase digests by two-dimensional polyacrylamide electrophoresis demonstrates that in the case of virus RNAs, the fingerprints thus obtained are very similar to those derived from uniformly labelled RNAs. The value of this technique is that it requires only 1-5 microgram of purified virus RNA and at least three orders of magnitude less radioactivity than is routinely employed in preparing uniformly labelled RNA.     

Comparison of the membrane interaction mechanism of two antimicrobial RNases: RNase 3/ECP and RNase 7

Eosinophil cationic protein (ECP/RNase 3) and the skin derived ribonuclease 7 (RNase 7) are members of the RNase A superfamily. RNase 3 is mainly expressed in eosinophils whereas RNase 7 is primarily secreted by keratinocytes. Both proteins present a broad-spectrum antimicrobial activity and their bactericidal mechanism is dependent on their membrane destabilizing capacities. Using phospholipid vesicles as membrane models, we have characterized the protein membrane association process. Confocal microscopy experiments using giant unilamellar vesicles illustrate the morphological changes of the liposome population. By labelling both lipid bilayers and proteins we have monitored the kinetic of the process. The differential protein ability to release the liposome aqueous content was evaluated together with the micellation and aggregation processes. A distinct morphology of the protein/lipid aggregates was visualized by transmission electron microscopy and the proteins overall secondary structure in a lipid microenvironment was assessed by FTIR. Interestingly, for both RNases the membrane interaction events take place in a different behaviour and timing: RNase 3 triggers first the vesicle aggregation, while RNase 7 induces leakage well before the aggregation step. Their distinct mechanism of action at the membrane level may reflect different in vivo antipathogen functions.     

Studies on the sequence of the 3'-terminal region of turnip-yellow-mosaic-virus RNA.

A fragment representing the 3'-terminal 'tRNA-like' region of turnip yellow mosaic (TYM) virus RNA has been purified following incubation of intact TYM virus RNA with Escherichia coli 'RNase P'. This fragment, which is 112+3-nucleotides long has been completely digested with T1 RNase and pancreatic RNase and all the oligonucleotides present in such digests have been sequenced using 32P-end labelling techniques in vitro. The TYM virus RNA fragment is free of modified nucleosides and does not contain a G-U-U-C-R sequence. Using nuclease P1 from Penicillium citrinum, the sequence of 26 nucleotides from the 5' end and 16 nucleotides from the 3' end of this fragment has been deduced. The nucleotide sequence at the 5' end of the TYM virus RNA fragment indicates that this fragment includes the end of the TYM virus coat protein gene.     

Study of RNA distribution in the nucleolar components of Ehrlich cell using RNase-gold method

The RNA distribution in Ehrlich tumour cell nucleoli has been investigated using RNase-gold method. This technique has been applied to sections of cells prepared under various fixation and embedding conditions. As expected, the specificity and intensity of labelling by gold particles have varied according to the experimental conditions used. Interestingly, however, it has been noted that the localization of gold particles does also vary and in particular within the fibrillar centre. This observation underlines the interest of assaying the RNase-gold complex under various conditions. The gold particles were particularly concentrated over the granular component and to a lesser extent, in the dense fibrillar component. In the latter constituent, it has been noted that the gold markers were preferentially localized at the edge of the dense fibrils. Surprisingly, a few gold particles have also been detected in the fibrillar centres. The weak labelling has persisted even after pepsin or DNase extraction but has completely disappeared after RNase extraction. Further, an inhibition of rRNA synthesis by a treatment with actinomycin D has not produced a significant decrease of the number of gold particles present in the fibrillar centre. These results suggest that fibrillar centres contain a small amount of RNA which would not correspond to pre-rRNA.     

Study of RNA distribution in the nucleolar components of Ehrlich cell using RNase-gold method

Summary The RNA distribution in Ehrlich tumour cell nucleoli has been investigated using RNase-gold method. This technique has been applied to sections of cells prepared under various fixation and embedding conditions. As expected, the specificity and intensity of labelling by gold particles have varied according to the experimental conditions used. Interestingly, however, it has been noted that the localization of gold particles does also vary and in particular within the fibrillar centre. This observation underlines the interest of assyying the RNase-gold complex under various conditions.The gold particles were particularly concentrated over the granular component and to a lesser extent, in the dense fibrillar component. In the latter constitutent, it has been noted that the gold markers were preferentially localized at the edge of the dense fibrils. Surprisingly, a few gold particles have also been detected in the fibrillar centres. The weak labelling has persisted even after pepsin or DNase extraction but has completely disappeared after RNase extraction. Further, an inhibition of rRNA synthesis by a treatment with actinomycin D has not produced a significant decrease of the number of gold particles present in the fibrillar centre. These results suggest that fibrillar centres contain a small amount of RNA which would not correspond to pre-rRNA.     

Effects of viscosity on phototactic movement and period of cell rotation in Cryptomonas sp.

The "protective" effect of polyamines and related metabolites on wounded storage tissues was investigated in two experimental systems: RNase activity of potato ( Solanum tuberosum L.) tuber discs, and betacyanin leakage from beet root ( Beta vulgaris L.) discs. Potato tuber discs evince a dramatic rise in RNase activity 24 h after excision, and this rise is completely blocked by the polyamines, spermidine and spermine. The effect is concentration-dependent, and several precursors of polyamines are also effective to various degrees in inhibiting the rise in RNase activity. Polyamines inhibit both the wound-induced RNase and the rise in RNase activity which may result from other senescence-linked events. Betacyanin leakage from beet root discs is enhanced upon aging of discs and in response to various treatments which may cause membrane damage. Spermidine and spermine effectively inhibit betacyanin leakage which results from aging at 25°C, from incubation at higher temperatures (35–40°C), from freezing of discs, or from treatment of discs with RNase or protease. It is suggested that polyamines decrease RNase activity and solute leakage in sliced storage tissues by affecting primary wound-induced destabilization of cell membranes.     

The nucleotide sequence at the 5'' end of foot and mouth disease virus RNA.

Foot and mouth disease virus RNA has been treated with RNase H in the presence of oligo (dG) specifically to digest the poly(C) tract which lies near the 5' end of the molecule (10). The short (S) fragment containing the 5' end of the RNA was separated from the remainder of the RNA (L fragment) by gel electrophoresis. RNA ligase mediated labelling of the 3' end of S fragment showed that the RNase H digestion gave rise to molecules that differed only in the number of cytidylic acid residues remaining at their 3' ends and did not leave the unique 3' end necessary for fast sequence analysis. As the 5' end of S fragment prepared form virus RNA is blocked by VPg, S fragment was prepared from virus specific messenger RNA which does not contain this protein. This RNA was labelled at the 5' end using polynucleotide kinase and the sequence of 70 nucleotides at the 5' end determined by partial enzyme digestion sequencing on polyacrylamide gels. Some of this sequence was confirmed from an analysis of the oligonucleotides derived by RNase T1 digestion of S fragment. The sequence obtained indicates that there is a stable hairpin loop at the 5' terminus of the RNA before an initiation codon 33 nucleotides from the 5' end. In addition, the RNase T1 analysis suggests that there are short repeated sequences in S fragment and that an eleven nucleotide inverted complementary repeat of a sequence near the 3' end of the RNA is present at the junction of S fragment and the poly(C) tract.     

Mutational analysis of the RNA component of Saccharomyces cerevisiae RNase MRP reveals distinct nuclear phenotypes

The 340-nucleotide RNA component of Saccharomyces cerevisiae RNase MRP is encoded by the single-copy essential gene, NME1. To gain additional insight into the proposed structure and functions of this endoribonuclease, we have extensively mutagenized the NME1 gene and characterized yeast strains expressing mutated forms of the RNA using a gene shuffle technique. Strains expressing each of 26 independent mutations in the RNase MRP RNA gene were characterized for their ability to grow at various temperatures and on various carbon sources, stability of the RNase MRP RNA and processing of the 5.8S rRNA (a nuclear function of RNase MRP). 11 of the mutations resulted in a lethal phenotype, six displayed temperature-conditional lethality, and several preferred a non-fermentable carbon source for growth. In those mutants that exhibited altered growth phenotypes, the severity of the growth defect was directly proportional to the severity of the 5.8S rRNA processing defect in the nucleus. Together this analysis has defined essential regions of the RNase MRP RNA and provides evidence that is consistent with the proposed function of the RNase MRP enzyme.     

Photooxidation and carbethoxylation of a minor ribonuclease from Aspergillus saitoi.

In order to investigate the nature of amino acid residues involved in the active in the active site of a ribonuclease from Aspergillus saitoi, the pH dependence of the rates of inactivation of RNase Ms by photooxidation and modification with diethylpyrocarbonate were studied. (1) RNase Ms was inactivated by illumination in the presence of methylene blue at various pH's. The pH dependence of the rate of photooxidative inactivation of RNase Ms indicated that at least one functional group having pKa 7.2 was involved in the active site. (2) Amino acid analyses of photooxidized RNase Ms at various stages of photooxidative inactivation at pH's 4.0 and 6.0 indicated that one histidine residue was related to the activity of RNase Ms, but that no tryptophan residue was involved in the active site. (3) 2',(3')-AMP prevented the photooxidative inactivation of RNase Ms. The results also indicated the presence of a histidine residue in the active site. (4) Modification of RNase Ms with diethylpyrocarbonate was studied at various pH's. The results indicated that a functional group having pKa 7.1 was involved in the active site of RNase Ms.     

Effect of ribonucleases on cell-mediated lympholysis reaction and on GM-CFC colonies in bone marrow culture

Natural dimer of bovine seminal ribonuclease (AS RNase) suppressed markedly DNA synthesis in allogeneic mixed lymphocyte culture (MLC) of normal human lymphocytes and simultaneously inhibited induction of cytotoxic effector cells within the sensitization phase of indirect cell-mediated lympholysis (CML) reaction. The last purification step of the AS RNase isolation procedure did not increase the suppressive activity of AS RNase compared to a less purified preparation (ZS RNase), thus, the later preparation was mostly used. ZS RNase (10 micrograms/ml) caused 50% inhibition of MLC reaction whereas pancreatic ribonuclease (A RNase) was 10 times less effective. The suppressive effect of RNases added in the beginning of the sensitization phase of the CML reaction correlated with that observed in the MLC reaction. The concentrations of ZS RNase (10 micrograms/ml), A RNase (100 micrograms/ml), and additionally tested cyclosporin A (0.5 microgram/ml) resulted in nearly total abrogation of cytolysis in CML. ZS RNase added after the sensitization of effector cells did not influence their cytolytic action on target cells within the destruction phase of CML. Natural killer and killer cell activities in normal peripheral lymphocytes were not inhibited by ZS RNase at the concentration of 330 micrograms/ml. ZS RNase (20 micrograms/ml), cocultivated 1 h with normal human bone marrow cells and then washed off, enhanced formation of GM-CFC colonies in semisolid agar culture up to 200%. Simultaneously tested antilymphocyte globulin increased the number of GM-CFC colonies at the average of 128%. This stimulating effect on colony formation appeared also in bone marrow culture of patients suffering with various hematological disorders. The possibility of utilizing the preparations gained from seminal plasma in clinical bone marrow transplantation is discussed.     

Studies on the restoration of the activities of Ribonucleases by polyamines in the presence of various ribonuclease inhibitors.

The effect of polyamines on ribonucleases in the presence of various inhibitors (poly(G), heparin, and rat liver RNase inhibitor) has been studied. Bovine pancreatic RNas A and a ribonuclease from horse submaxillary gland (RNase HS) were inhibited by the inhibitors, but RNase T1 and RNase M were not inhibited. Polyamines were found to restore the activites of RNase A and RNase HS inhibited by poly(G) or heparin but not those activities inhibited by rat liver RNase inhibitor. When poly(U) and poly(C) were used as substrates, the inhibitory effects of poly(G) and heparin were greater with poly(U) than poly(C) as a substrate. However, when poly(C) was used as a substrate in the presence of either of the above inhibitors, the restoration of RNase activity by sperimine was more efficient. In fact, a stimulatory effect was observed. From the double-reciprocal plots, it was concluded that polyamines restored the activiities of RNases by increasing the availability of the substrate and enzyme to each other. The restoration of enzyme activity by polyamines occurred through the binding of the polyamines to the inhibitor and the subsequent release of enzyme from the inhibitor.     

Studies on the state of tyrosyl residues in a ribonuclease from seminal vesicles.

In order to study the state of tyrosyl residues in a ribouuclease from bovine semina vesicles [EC 3.1.4.22, RNase Vs1] several lines of experiments were carried out. Spectrophotometric titration of RNase Vs1 indicated that two out of 8 tyrosine residues were titrated very easily and their apparent pKa values were about 9.8. Next, about 4 residues were titrated at pH up to 13.5. The remaining 2 residues were titrated time-dependently at pH 13.5. In 8 M urea, about 6 tyrosine residues were titrated with apparent pK4 values of about 11.2 and about 2 residues were titrated time-dependently at pH 13.5. Acetylation of RNase Vs1 with N-acetylimidazole was studied at pH 7.5. In aqueous solution, about 1.1-3.5 tyrosine residues were acetylated, depending on the experimental conditions, and in 8 M urea, 5.3 tyrosine residues were modified. RNase Vs1 was nitrated with tetranitromethane at pH 7.5. In aqueous solution, about 2.5 tyrosine residues were nitrated very easily; the enzymatic activity of the modified enzymes was 130-200% of that of the native enzyme. In 8 M urea, the reactivity of the tyrosine residues increased and about 4-5.5 residues were modified. The results of chemical modification and spectrophotometric titration indicated that about two tyrosine residues in RNase Vs1 were exposed to the solvent and were more reactive to various reagents, and 3-4 tyrosine residues were less reactive. The final 2 residues were not accessible to the reagent even in the presence of urea, but were titraten at pH 13.5. The solvent perturbation difference spectrum using ethylene glycol as a perturbant indicated that about 4 tyrosine residues were perturbed. When the pH of the enzyme solution was changed from 7.0 to 1.0, the change in optical density of RNase Vs1 due to denaturation blue shift was about 1,600 at 287nm. The optical density change at 287 nm of native RNase Vs1 on exposure to 8 M urea and 6 M guanidine-HCl indicated that the environments of 2-3 and 4 tyrosine residues were changed by the addition of the denaturants, urea and guanidine-HCl, respectively. In RNase Vs1 having about four nitrotyrosine residues, the two most inaccessible tyrosine residues remained resistant to titration with alkali. On adding nucleotide, nitrated RNase Vs1 gave a difference spectrum in the ultraviolet region but not in 320-460 nm region, where nitrotyrosine residues absorb light. This may indicate that tyrosine residues located relatively near the surface of the molecule are not perturbed directly by nucleotide binding.     

Phylogenetic comparative mutational analysis of the base-pairing between RNase P RNA and its substrate.

We have studied the base-pairing between the 3'-terminal CCA motif of a tRNA precursor and RNase P RNA by a phylogenetic mutational comparative approach. Thus, various derivatives of the Escherichia coli tRNA(Ser)Su1 precursor harboring all possible substitutions at either the first or the second C of the 3'-terminal CCA motif were generated. Cleavage site selection on these precursors was studied using mutant variants of M1 RNA, the catalytic subunit of E. coli RNase P, carrying changes at positions 292 or 293, which are involved in the interaction with the 3'-terminal CCA motif. From our data we conclude that these two C's in the substrate interact with the well-conserved G292 and G293 through canonical Watson-Crick base-pairing. Cleavage performed using reconstituted holoenzyme complexes suggests that this interaction also occurs in the presence of the C5 protein. Furthermore, we studied the interaction using various derivatives of RNase P RNAs from Mycoplasma hyopneumoniae and Mycobacterium tuberculosis. Our results suggest that the base-pairing between the 3'-terminal CCA motif and RNase P is present also in other bacterial RNase P-substrate complexes and is not limited to a particular bacterial species.     

Nucleotide sequences adjacent to the proteins covalently linked to the cowpea mosaic virus genome. Sequence determination after labelling in vitro using RNA ligase.

The sequences of the first 17 nucleotides of cowpea mosaic virus middle and bottom RNAs adjacent to the covalently-linked proteins have been determined. Sequences of the oligonucleotides, produced by complete T1 RNase digestion, were established after labelling of the 3' termini in vitro using RNA ligase. Both sequences are A/U-rich, the first nine nucleotides being identical.     

Ethylenediamine derivatives efficiently react with oxidized RNA 3′ ends providing access to mono and dually labelled RNA probes for enzymatic assays and in vivo translation

Development of RNA-based technologies relies on the ability to detect, manipulate, and modify RNA. Efficient, selective and scalable covalent modification of long RNA molecules remains a challenge. We report a chemical method for modification of RNA 3′-end based on previously unrecognized superior reactivity of N-substituted ethylenediamines in reductive amination of periodate-oxidized RNA. Using this method, we obtained fluorescently labelled or biotinylated RNAs varying in length (from 3 to 2000 nt) and carrying different 5′ ends (including m⁷G cap) in high yields (70–100% by HPLC). The method is scalable (up to sub-milligrams of mRNA) and combined with label-facilitated HPLC purification yields highly homogeneous products. The combination of 3′-end labelling with 5′-end labelling by strain-promoted azide-alkyne cycloaddition (SPAAC) afforded a one-pot protocol for site-specific RNA bifunctionalization, providing access to two-colour fluorescent RNA probes. These probes exhibited fluorescence resonance energy transfer (FRET), which enabled real-time monitoring of several RNA hydrolase activities (RNase A, RNase T1, RNase R, Dcp1/2, and RNase H). Dually labelled mRNAs were efficiently translated in cultured cells and in zebrafish embryos, which combined with their detectability by fluorescent methods and scalability of the synthesis, opens new avenues for the investigation of mRNA metabolism and the fate of mRNA-based therapeutics.     

A new fluorescence-based, hydrophobic photolabeling technique for analyzing membrane-associated proteins

We introduce a new, fluorescent and photoactivatable fatty acid derivative (SANU) for hydrophobic labelling of membrane-bound proteins. The technique allows fast and highly sensitive screening of hydrophobically inserting proteins analyzed by SDS-PAGE with a detection limit below 0.1 pmol. A reliable calculation of labelling efficiencies is achieved by simultaneous densitometry of fluorescence and protein staining. We have applied the new technique on the membrane inserting protein talin, G-actin, and, as a negative control, on RNase, which only binds electrostatically to negatively charged lipid interfaces. In several ways superior to radiolabelling, we can recommend this technique for all laboratories under any circumstances.     

Expression of RNase Rh from Rhizopus niveus in yeast and characterization of the secreted proteins.

The full-length cDNA encoding RNase Rh, which is secreted extracellularly by Rhizopus niveus, was isolated and its nucleotide sequence was determined. It was placed under control of the promoter of the glyceraldehyde 3-phosphate dehydrogenase gene of Saccharomyces cerevisiae in a high expression vector in yeast. Since yeast cells transformed by this plasmid poorly secreted RNase into the medium, the plasmid pYE RNAP-Rh was constructed, in which the signal sequence of RNase Rh was replaced by the prepro-sequence of aspartic proteinase-I, one of the extracellular enzymes secreted by R. niveus. Yeast cells harboring pYE RNAP-Rh produced RNase efficiently (ca. 40 micrograms/ml) into the medium. The product was a mixture of six enzymes (RNase RNAP-Rhs) having 3, 5, 9, 13, 14, and 16 additional amino acid residues attached to the amino terminus of the mature RNase Rh. The major product was the RNase with three additional amino acids at the amino terminus. Limited digestion of RNase RNAP-Rhs with staphylococcal V8 protease succeeded in shortening the various lengths of extra amino acid residues attached to the amino terminus of RNase Rh, yielding an RNase that has 3 additional amino acids at the amino terminus. It has been named RNase RNAP-Rh. The RNase RNAP-Rh showed the same specific activity and CD spectra as those of RNase Rh, suggesting that the two have similar conformations to each other around aromatic amino acid residues and the peptide backbone.     

Spectrin-like proteins in plant nuclei

We analysed the presence and localization of spectrin-like proteins in nuclei of various plant tissues, using several anti-erythrocyte spectrin antibodies on isolated pea nuclei and nuclei in cells. Western blots of extracted purified pea nuclei show a cross-reactive pair of bands at 220-240 kDa, typical for human erythrocyte spectrin, and a prominent 60 kDa band. Immunolocalization by means of confocal laser scanning microscopy reveals spectrin-like proteins in distinct spots equally distributed in the nucleoplasm and over the nuclear periphery, independent of the origin of the anti-spectrin antibodies used. In some nuclei tracks of spectrin-like proteins are also observed. No signal is present in nucleoli. The amount and intensity of signal increases when nuclei were extracted, successively, with detergents, DNase I and RNase A, and high salt, indicating that the spectrin-like protein is associated with the nuclear matrix. The labelling is similar in nuclei of various plant tissues. These data are the first that show the presence and localization of spectrin-like epitopes in plant nuclei, where they may stabilize specific interchromatin domains.