The specific endoribonuclease activity of small nuclear and cytoplasmic alpha-RNPs

For the first time small nuclear ribonucleoprotein particles (alpha-RNP) tightly bound to chromatin as well as cytoplasmic alpha-RNP are shown to possess strong and regulated endonuclease activity specific for mRNAs and hnRNAs. The enzymatic nature of this activity is confirmed, and the optimal conditions detected. This RNase activity is controlled by the action of a differentiating stimulus, dimethylsulfoxide, in human K562 cells. Small alpha-RNP involvement in the coordinated control of stability of pre-messenger RNA and messenger RNA molecules is suggested.     

Re-expression of various i-antigens in Dileptus anser after temporary transformation of serotype

RNP particles containing 20S prosomes (alpha RNP) isolated from human epidermoid carcinoma cell line A-431 are shown to posses strong and regulated endonuclease activity specific for high-molecular-weight RNA, particularly, specific mRNAs. Furthermore, alpha-RNP destabilize the 3'-untranslated regions of c-myc mRNA, creating a specific cleavage pattern. Cleavage point within Alu sequence in high-molecular-weight RNA has been localized by primer-extension method. This RNase activity is induced under the action of EGF. alpha-RNP involvement in the coordinated control of processing and stability of specific messenger RNA molecules is suggested. The endoribonuclease activity of alpha-RNP can represent a link between EGF signalling pathway and RNA processing and degradation.     

Selective effect of inductors of apoptosis on the endoribonuclease activity of 26S proteasomes and alpha-RNP particles in K562 cells: possible involvement of 26S proteasomes and alpha-RNP in the regulation of RNA stability

It has been shown that endoribonuclease activity of alpha-RNP particles and 26S proteasomes are changed under the action of inductors of programmed cell death. Treatment of K562 cells with inductors of apoptosis--doxorubicin (adriamycin) and diethylmaleate--lead to a significant stimulation of RNAse activity of alpha-RNP and to reduction of proteasome RNase activity. The enzymatic activity under study has been shown to be specifically and selectively dependent on phosphorylation of subunits of alpha-RNP particles and 26S proteasomes. The characteristics of RNAse activity of different subpopulations of proteasomes differ. The specificity of a subpopulation of proteasomes exported from the cell has been demonstrated. Proteasome and alpha-RNP involvement in the coordinated control of stability of various specific messenger RNA molecules is suggested, and one of the mechanisms of this control might be the export of specific subpopulation of proteasomes from the cell.     

Phosphorylation of proteasomes and alpha-RNP from rat liver cells

In eukaryotic cells the population of proteasomes is heterogeneous. Here we have shown that proteasomes from nuclei and cytoplasm of rat liver cells differ in their subunit patterns. The subunit pattern of alpha-RNP differs from that of proteasomes, however, alpha-RNP particles contain the number of 26S proteasome subunits. Moreover, the proteasomes contain subunits of alpha-RNP. We have shown for the first time that nuclear proteasomes and alpha-RNP are hyperphosphorylated on threonine residues. Differences in phosphorylation state of subunits of nuclear and cytoplasmic proteasomes and alpha-RNP on threonine and tyrosine residues have been revealed. A suggestion is put forward that hyperphosphorylation of subunits may determine nuclear localization of these complexes in rat liver cells. The results obtained suggest that a highly specialized system of protein kinases and phosphatases may be involved in the regulation of phosphorylation state of different populations of proteasomes and alpha-RNP in rat liver cells.     

Cortisone-induced small RNP tightly bound to chromatin

The small nuclear RNP (alpha-RNP) tightly bound to chromatin has been isolated. alpha-RNP can be removed from chromatin together with the acid-soluble proteins. The RNA from this RNP has been isolated; its electrophoretic mobility is equal to that of 4 S RNA. The study of the resistance of alpha-RNA to RNases (A, T1 and S1) in salt solutions of various ionic strengths allows us to conclude that the alpha-RNA has a well-developed secondary structure. The alpha-RNA is tightly associated with the protein moiety of alpha-RNP and has developed secondary structure. The alpha-RNA is tightly associated with the protein moiety of alpha-RNP and has a high metabolic activity.     

EGF-dependent association of 20S-proteasome and alpha-RNP particles with the epidermal growth factor receptor in A-431 cells

Here we demonstrate that the epidermal growth factor (EGF) induces association of prosomes (20S-proteasomes) with its receptor in A-431 cells. Additionally, ligand-dependent association of ribonucleoprotein particles (alpha-RNP), containing small ALU-like RNA, with the EGF receptor was demonstrated. A suggestion has been put forward on the involvement of prosomes and alpha-RNP in the EGF signal transmission to different stages of gene expression.     

Alu-DNA repeat-binding protein p68 is a part of Alu-RNA containing alpha-RNP.

An Alu-DNA repeat-binding protein with a molecular mass of 68 kDa (p68) is identified in the somatic human cell nucleoplasm. Gel mobility shift assay (GMSA), South-western blotting and affinity purification on DNA attached to the carrier were used in the identification. GMSA revealed multiple complexes with the exponential dependence of their relative mobility. A narrow binding site of the p68 was revealed using synthetic oligonucleotides. It is located between the A-box and B-box of the RNA polymerase III promoter and is identical to that reported for the Alu-binding protein from human spermatozoids. The same narrow binding site, the similarity of the isolation procedure from germ and somatic cells, and similar binding properties and molecular masses suggest homology of the two proteins. Antibodies raised against Alu-protein complexes led to hypershift of the complexes in GMSA and stained p68 in active fractions in human spermatozoids and in Alu-RNA-containing alpha-RNP particles. Immunofluorescence of a HeLa cell monolayer revealed an intranuclear dot pattern with the dots corresponding to euchromatin areas and some dots located at the cell periphery in the cytoplasm. alpha-RNP particles bound Alu-DNA in vitro and contained p68 as shown using the immunogold procedure. Alu-DNA binding activity was revealed in cytoplasm as well as in nucleoplasm. The possible nature of the main Alu-DNA binding protein and its involvement in the particle structure are discussed.     

The small RNP acceptor of glucocorticoids bound to chromatin in liver cell nuclei

The buoyant density in the CsCl gradient of the small nuclear RNP tightly bound to chromatin has been studied. It was shown that the buoyant density of alpha-RNP is characteristic for ribonucleoproteins (p = 1.36-1.50 g/cm3). The alpha-particles are of extraordinary stability. These RNP were shown to remain stable under drastic conditions (high ionic strength, SDS, 6 M urea) and resist unfixed caesium chloride density centrifugation. The alpha-RNA hybridizes with total rat liver DNA at C0t1/2 = 10(3). The oligonucleotide analysis of the alpha-RNA shows that the alpha-RNA is heterogeneous.     

Lack of peptide-release activity responding to codon UGA in Mycoplasma capricolum.

In Mycoplasma capricolum, a relative of Gram-positive eubacteria with a high genomic AT-content (75%), codon UGA is assigned to tryptophan instead of termination signal. Thus, in this bacterium the release factor 2 (RF-2), that recognizes UAA and UGA termination codons in eubacteria such as Escherichia coli and Bacillus subtilis, would be either specific to UAA or deleted. To test this, we have constructed a cell-free translation system using synthetic mRNA including codon UAA [mRNA(UAA)], UAG [mRNA(UAG)] and UGA [mRNA(UGA)] in-frame. In the absence of tryptophan, the translation of mRNA(UGA) ceased at UGA sites without appreciable release of the synthesized peptides from the ribosomes, whereas with mRNA(UAA) or mRNA(UAG) the bulk of the peptides was released. Upon addition of the E.coli S-100 fraction or B.subtilis S-100 fraction to the translation system, the synthesized peptides with mRNA(UGA) were almost completely released from the ribosomes, presumably because of the presence of RF-2 active to UGA in the added S-100 fraction. These data suggest that RF-2 is deleted or its activity to UGA is strongly weakened in M.capricolum.     

Translational activity of messenger ribonucleic acid isolated from unstimulated and phytohaemagglutinin-activated lymphocytes.

Purified cytoplasmic poly(A)+ RNA isolated from unstimulated pig lymphocytes has the same ability to direct translation in a range of cell-free systems as the corresponding mRNA from 20h phytohaemagglutinin-activated lymphocytes. Additional methylation of the mRNA is not required for maximum protein synthesis in the wheat-germ cell-free system. Misleading results are obtained if the mRNA preparations used are not adequately purified, and a method suitable for routine assessment of the degree of purification achieved is described. Cell-free protein-synthesizing systems from unstimulated lymphocytes translate added lymphocyte mRNA with lower efficiency than do comparable systems from phytohaemagglutinin-activated lymphocytes, whatever the source of the mRNA used.     

Differential regulation of MyoD and myogenin mRNA levels by nerve induced muscle activity.

The levels of mRNAs coding for the myogenic factors MyoD and myogenin were measured during synapse formation in developing muscle and in adult muscle, after denervation and reinnervation and after muscle paralysis induced by blocking of neuromuscular transmission by neurotoxins known to alter the density and localization of synaptic proteins such as the acetylcholine receptor (AChR). The mRNA levels of both factors depend on usage of the neuromuscular synapses, but they change to different extents. Myogenin mRNA levels decrease drastically with innervation and increase strongly following blocking of transmission whereas the level of MyoD mRNA showed only a small decrease in response to innervation, denervation or muscle paralysis by neurotoxins. Neither mRNA showed a synapse-related cellular distribution. The results suggest that nerve-induced electrical muscle activity determines the cellular ratio of MyoD and myogenin mRNAs in adult muscle.     

Hammerhead ribozymes to modulate telomerase activity of endometrial carcinoma cells.

Telomerase is an excellent target molecule for cancer therapy, though any effective agents have never been developed in human subjects. We designed a variety of hammerhead ribozymes against human telomerase RNA (hTR) and hTERT mRNA and studied their possibility as a tool for cancer therapy. To search promising target site of hTR, the catalytic actiuity of 3 kinds of hammerhead ribozymes was studied in cell-free system. They showed equivalent catalytic activity, but only 36-ribozyme, which was designed to cleave the template region of hTR, revealed telomerase inhibitory activity in an endometrial carcinoma cell line. Among hTERT-mRNA-targeted ribozymes, the ribozyme to cleave 13 nucleotides downstream from the 5'-end of hTERT mRNA (13-ribozyme) exhibited the strongest telomerase-inhibitory activity, and the ribozyme to cleave 59 nucleotides upstream from the poly(A) tail showed clear activity. Stable transfection studies confirmed that the 36-ribozyme as well as the 13-ribozyme suppressed telomerase. These observations suggest that the template region of hTR and 5'end of hTERT mRNA are promising target sites for ribozymes to reduce telomerase activity.     

Down-regulation of hepatic lipase gene expression and activity by fenofibrate.

The influence of the hypolipidemic drug, fenofibrate, on hepatic lipase (HL) gene expression and activity was investigated in the rat. Fenofibrate treatment provoked a dose-dependent decrease in HL mRNA levels. At a dose of 0.5% (w/w), HL mRNA levels were reduced to nearly 50% the levels in untreated controls. This decrease was parallelled by a comparable reduction in liver HL activity. The decrease in HL mRNA levels was already observed after 1 day of fenofibrate treatment. Whole liver perfusion experiments showed that the heparin-releasable HL activity in fenofibrate-treated livers dropped to 10% the activity in control livers. In conclusion, treatment with fenofibrate decreases HL gene expression, leading to a lowered activity of endothelium bound HL in fenofibrate-treated livers.     

Rigid structural requirement of the 5' terminus of mRNA for translational activity.

Oxidation by sodium periodate of the ribose cis-diol of the 5′ terminal of liver mRNA to the corresponding dialdehyde virtually destroyed its template activity in the wheat germ translation system. The rigid structural requirement for the ribose cis-diol is indicated by the failure of reduction of the dialdehyde to the corresponding primary alcohols to restore the template activity of the mRNA. Sodium periodate alone inhibited the translational system at concentrations above 0.25 mM. Purification of the periodate oxidized mRNA by sucrose density gradient centrifugation or exclusion on Sephadex G-100 did not increase its template activity. Periodate oxidized mRNA was not inhibitory to translation of untreated mRNA.     

Dynamics of denitrification activity of Paracoccus denitrificans in continuous culture during aerobic-anaerobic changes.

Induction and repression of denitrification activity were studied in a continuous culture of Paracoccus denitrificans during changes from aerobic to anaerobic growth conditions and vice versa. The denitrification activity of the cells was monitored by measuring the formation of denitrification products (nitrite, nitric oxide, nitrous oxide, and dinitrogen), individual mRNA levels for the nitrate, nitrite, and nitrous oxide reductases, and the concentration of the nitrite reductase enzyme with polyclonal antibodies against the cd1-type nitrite reductase. On a change from aerobic to anaerobic respiration, the culture entered an unstable transition phase during which the denitrification pathway became induced. The onset of this phase was formed by a 15- to 45-fold increase of the mRNA levels for the individual denitrification enzymes. All mRNAs accumulated during a short period, after which their overall concentration declined to reach a stable value slightly higher than that observed under aerobic steady-state conditions. Interestingly, the first mRNAs to be formed were those for nitrate and nitrous oxide reductase. The nitrite reductase mRNA appeared significantly later, suggesting different modes of regulation for the three genes. Unlike the mRNA levels, the level of the nitrite reductase protein increased slowly during the anaerobic period, reaching a stable value about 30 h after the switch. All denitrification intermediates could be observed transiently, but when the new anaerobic steady state was reached, dinitrogen was the main product. When the anaerobic cultures were switched back to aerobic respiration, denitrification of the cells stopped at once, although sufficient nitrite reductase was still present. We could observe that the mRNA levels for the individual denitrification enzymes decreased slightly to their aerobic, uninduced levels. The nitrite reductase protein was not actively degraded during the aerobic period.     

Activation of 2'',5''-oligoadenylate synthetase activity on induction of HL-60 leukemia cell differentiation.

A 27-fold increase in 2',5'-oligoadenylate synthetase activity, an enzyme associated with the antiproliferative actions of interferon (IFN), was observed after treatment of HL-60 human leukemia cells with dimethyl sulfoxide (DMSO), an inducer of granulocytic differentiation of the cells. Enzyme activity was elevated after 24 h of exposure to DMSO, was maximal at 48 hours, and declined thereafter. A comparable increase was observed after treatment with 1 U of alpha interferon (IFN-alpha) per ml or 8 U of beta interferon (IFN-beta) per ml. Elevated levels of expression of other IFN-inducible genes, including type I histocompatibility antigen (HLA-B) mRNA and 2',5'-oligoadenylate phosphodiesterase activity, were also observed with DMSO treatment. DMSO-treated HL-60 cells had an increased amount of a 1.8-kilobase mRNA for oligoadenylate [oligo(A)] synthetase when compared with that of control cells; both DMSO- and IFN-treated HL-60 cells also expressed 1.6-, 3.4-, and 4.3-kilobase mRNA. The increase in both oligo(A) synthetase activity and mRNA levels was inhibited by polyclonal antiserum to human IFN-alpha; however, no IFN-alpha mRNA could be detected in the cells. Antiserum to IFN-beta or gamma interferon (IFN-gamma) had no effect on oligo(A) synthetase expression or activity nor was there any detectable IFN-beta 1 or IFN-beta 2 mRNA in the cells. The anti-IFN-alpha serum did not block the elevation of HLA-B mRNA in DMSO-treated cells. These observations suggest that the increased expression of oligo(A) synthetase in DMSO-treated cells may be mediated by the release of an IFN-alpha-like factor; however, the levels of any IFN-alpha mRNA produced in the cells were extremely low.