Cleavage Of Ribosomal RNA Research Articles

Independent regulation of ppp(A2'p)nA-dependent RNase in NIH 3T3, clone 1 cells by growth arrest and interferon treatment.

The regulation of ppp(A2'p)nA-(2-5A)-dependent RNase (RNase L or RNase F) was investigated in NIH 3T3, clone 1 cells using 2-5A-binding and nuclease activity assays. Minimal levels of 2-5A-dependent RNase were detected in actively dividing clone 1 cells; these levels were independently induced by growth arrest or interferon treatment. Accordingly, levels of the RNase were enhanced during growth arrest by confluency regardless of the presence or absence of interferon or antibody to interferon in the media. Measurement of 2-5A-dependent RNase was unaffected by the addition of any of six different proteinase inhibitors to the cells prior to extraction. The expression of 2-5A-dependent RNase in growth-arrested, interferon-treated cells was still relatively low (about one-third to one-half of that found in similarly treated murine Ehrlich ascites tumor cells). Although this amount of 2-5A-dependent RNase could not be detected by 2-5A-mediated ribosomal RNA cleavage, the activity was identified using a more sensitive novel assay for 2-5A-dependent RNase. In addition, introduction of 2-5A or poly(I) X poly(C) into growth-arrested, interferon-treated cells resulted in some inhibition of protein synthesis. The results indicated that the expression of 2-5A-dependent RNase in NIH 3T3, clone 1 cells is regulated under different physiological conditions and that low levels of 2-5A-dependent RNase were insufficient to significantly inhibit encephalomyocarditis virus replication.

Cordycepin analogs of ppp5'A2‘p5'A2‘p5'A (2-5A) inhibit protein synthesis through activation of the 2-5A-dependent endonuclease.

Analogs of the triphosphate 2'-5'-linked adenylate trimer (ppp5'A2'p5'A2'p5'A, called 2-5A) which contain 3'-deoxyadenosine (cordycepin) instead of adenosine either in positions one and two, or in all three positions, are 10-100-fold less potent than is parent 2-5A in inhibition of protein synthesis in intact cells, when utilizing calcium co-precipitation techniques to introduce the 5'-triphosphate oligonucleotides into the cells. That the inhibition of protein synthesis was a consequence of activation of the 2-5A-dependent endonuclease by the 3'-deoxyadenosine analogs of 2-5A was demonstrated in obtaining the ribosomal RNA cleavage pattern that is characteristic of endonuclease activation by parent 2-5A. Additional results (i.e. lack of activity by the dimer species ppp5'(3'dA)2'p5'-(3'dA) or the monomer 3'dA) as well as kinetic analysis both in intact cells and in cell-free extracts provided further evidence that the inhibition of protein synthesis observed with these 3'-deoxyadenosine 2-5A analogs was not due to their degradation to the antimetabolite monomer unit 3'-deoxyadenosine.

Interferon-induced synthesis of 2-5A-dependent RNase in mouse JLS-V9R cells

Interferon treatment of mouse JLS-V9R cells resulted in a 10- to 20-fold increase in the levels of the 2–5A (ppp(A2′p) n A)-dependent RNase. The nuclease was monitored in cell extracts by covalent and noncovalent binding of 32P-labeled 2–5A derivatives to the nuclease and by the appearance of 2–5A-mediated ribosomal RNA cleavage products. Untreated JLS-V9R cells contained very low levels of the nuclease (5–10% of that found in control Ehrlich ascites tumor cells). An increase in the nuclease was first detected between 3 and 6 hr after interferon treatment and was completely inhibited by actinomycin D. Control experiments indicated that the increase in the nuclease was not due to an intracellular redistribution of the enzyme. The results indicate that in JLS-V9R cells, interferon controls the activity of the 2–5A system at two levels: by inducing the synthesis of both the 2–5A-dependent RNase and the 2–5A-synthetase.

Dephosphorylated Core of 2′-5′ Oligoadenylate Exerts Its Antimitogenic Effect through Mechanisms Different from the 2′-5′A-Dependent Endonuclease

The 5′ terminally-dephosphorylated "core" of the 2′-5′ linked adenylate oligomer ppp (A2′p)2A, termed 2-5A core [(A2′p)2A], exerts an antimitogenic effect when added to serum-stimulated 3T3 fibroblasts. We now report that this inhibition of DNA synthesis is not an indirect effect resulting from ppp(A2′p)2A-dependent endonuclease activation. We have shown that the 2-5A core does not activate the ppp(A2′p)2A-dependent endonuclease when added to intact cells, based on (1) lack of the specific ribosomal RNA cleavage characteristic of the ppp(A2′p)2A-dependent endonuclease, and (2) antimitogenic activity in a cell line (NIH-3T3, clone 1) which is defective in its ability to show activation of the ppp(A2′p)2A-dependent endonuclease.

The ppp(A2'p)nA and protein kinase systems in wild-type and interferon-resistant Daudi cells.

Daudi cells, a human lymphoblastoid line, are exceptionally sensitive to the growth inhibitory effects of interferon, 1 unit/ml being sufficient to inhibit cell growth. In addition, interferon treatment of these cells severely inhibits the incorporation of exogenous thymidine into DNA and causes cells to accumulate in the G1(G0) at the expense of the S phase of the cell cycle. The possible involvement of ppp(A2'p)nA(n = 2 to less than or equal to 4) in these effects has been investigated. No (less than 1 nM) ppp(A2'p)nA or (A2'p)nA or alternative products of the ppp(A2'p)nA synthetase [e.g. NAD (2'pA)2] were detected in interferon-treated cells. In addition no evidence was obtained for the occurrence of ppp(A2'p)nA-mediated ribosomal RNA cleavage in these cells even after several days of treatment with relatively high doses of interferon. A line of Daudi cells which is resistant to all three of the above effects of interferon was selected. The wild type and resistant lines were compared with respect to the ppp(A2'p)nA and interferon and double-stranded RNA (dsRNA)-mediated protein kinase systems. The resistant line was not receptor-negative as it responded to interferon by the production of elevated levels of the ppp(A2'p)nA synthetase similar to those observed in extracts from wild-type cells. There was no detectable difference between the lines in the levels of the (2'-5')phosphodiesterase responsible for the degradation of ppp(A2'p)nA. There was, however, about a twofold increase in the ppp(A2'p)nA-dependent endoribonuclease activity in response to interferon with extracts from the wild-type but not the resistant cells. In addition, although the dsRNA-dependent protein kinase activity increased in both types of cell there was a striking reduction in the level of protein phosphorylation in general in response to interferon with material from the wild-type but not the resistant cells.

Control of the ppp(a2'p)nA system in HeLa cells. Effects of interferon and virus infection.

HeLa cells have an unusually high level of ppp(A2'p)nA synthetase (n = 2 to greater than or equal to 4) even in the absence of interferon treatment. In accord with this ppp(A2'p)nA and ppp(A2'p)nA-mediated ribosomal RNA cleavage occur naturally in response to encephalomyocarditis virus infection in control as well as in interferon-treated cells. Despite this, in the absence of interferon treatment, encephalomyocarditis virus grows well in these cells. A possible explanation for this paradox is that the ppp(A2'p)nA dependent RNase is lost or inactivated at later times post-infection in control but not in interferon-treated cells. It appears, therefore, to be the prevention by interferon of the virus-mediated inhibition of the ppp(A2'p)n-dependent nuclease rather than the absolute level or induction of the ppp(A2'p)nA synthetase which is crucial for the activity of the ppp(A2'p)nA system in HeLa cells. These results provide evidence for a further level of control in the ppp(A2'p)nA system and show that limited ppp(A2'p)nA-mediated ribosomal RNA cleavage alone is not sufficient to cause an inhibition of virus growth.

Ribosomal RNA cleavage, nuclease activation and 2-5A(ppp(A2'p)nA) in interferon-treated cells.

Ribosomal RNA (rRNA) in intact ribosomes is cleaved into discrete products on incubation of reticulocyte lysates or L-cell extracts with ppp(A2'p)3A. Cleavage of rRNA may, therefore, provide a useful assay for 2-5A (ppp)A2'p)nA; n = 2 to 4) or for the presence of a 2-5A-dependent nuclease. The results with reticulocyte lysates differed from those obtained in the L-cell-free system in that (a) a different RNA cleavage pattern was produced (with added L-cell ribosomes) and (b) cleavage was fully activated by the analogue ppp(A2'p)3A3'pCp. As might be expected from the relatively high levels of 2-5A present in interferon-treated, encephalomyocarditis virus (EMC)-infected L-cells, rRNA extracted from these cells was also cleaved. The cleavage pattern observed overlapped with that obtained on incubation of an L-cell-free system with 2-5A. Thus, not only is 2-5A present, but the 2-5A-dependent nuclease also appears to be active, in interferon-treated, EMC-infected L-cells.

Multiple ribosomal RNA cleavage pathways in mammalian cells.

The sequence content of mouse L cell pre-rRNA was examined by RNA gel transfer and blot hybridization. Nuclear RNAs were separated by agarose gel electrophoresis, transferred to diazo-paper, and hybridized to twelve different restriction fragments that are complementary to various sections of 45S pre-rRNA. An abundant new 34S pre-rRNA and less abundant new 37S, 26S and 17S pre-rRNAs were detected. The presence of these new pre-rRNAs suggests the existence of at least two new pre-rRNA cleavage pathways. 34S and 26S pre-rRNAs were also detected in HeLa cells suggesting that these new cleavage pathways are characteristic of mammalian cells. Further, an abundant new 12S precursor to 5.8S rRNA was also detected and is common to all the proposed cleavage pathways. The previously identified 45S, 41S, 32S and 20S pre-rRNAs were readily detected and their general structure confirmed. The 20S pre-rRNA is characteristic of the known pathway used by HeLa and other cells, and its presence suggests that growing mouse L cells use this pre-rRNA cleavage pathway. The 36S pre-rRNA characteristic of the previously described mouse L cell cleavage pathway was not detected. In all these cleavage pathways pre-rRNA cleavage sites are apparently identical and occur at or near the termini of the mature 18S, 5.8S and 28S rRNA sequences. The pathways differ only in the temporal order of cleavage at these sites. The position of the 5.8S rRNA sequence was located within the internal transcribed spacer. The known and conserved sequence of 5.8S rRNA from several organisms predicts a characteristic pattern of restriction enzyme sites for 5.8S rDNA. Internal transcribed spacer rDNA was mapped with restriction enzymes, and the characteristic pattern was found near the midpoint of the internal transcribed spacer. This places the 5.8S rRNA sequence at or near the 5' terminus of 32S pre-rRNA.

Interaction between membrane functions and protein synthesis in reticulocytes: specific cleavage of 28-S ribosomal RNA by a membrane constituent.

A factor isolated from rabbit reticulocyte white ghosts by Triton X-100 treatment blocks protein synthesis at the elongation-termination stage. Factor-treated ribosomes were found to have an identical buoyant density to that of control ribosomes. When incubated with either reticulocyte ribosomes or ribosomal RNA, the factor products specific cuts in the 28-S ribosomal RNA compenent without damaging the 18-S RNA. Incubations of pancreatic or T1 RNase, with ribosomal RNA, at similar protein-synthesis inhibitory concentrations effected a complete breakdown to oligo and mononucleotides. When challenged with isolated 28-S or 18-S reticulocyte ribosomal RNA, the highly purified factor only attacked the 28-S RNA species. There was no accumulation of nucleotides or oligonucleotides and we concluded that the membrane factor causes inhibition of protein synthesis by having a specific endonucleolytic cleavage activity.

Specific cleavage of ribosomal RNA caused by alpha sarcin

Alpha sarcin causes the specific cleavage of RNA from 80S ribosomes and 60S subunits of yeast, but not from the 40S subunits to produce a small RNA fragment. The fragment was also produced on treatment of the 60S subunits of wheat germ ribosomes. The fragment has a molecular weight of 100,000 and is a cleavage product of the large RNA species in the 60S subunits. The fragment is not derived from the 5'end of the yeast 25S RNA nor does it bind to 5.8S RNA and we propose that the fragment represents the 3' terminal 320 nucleotides of 25S rRNA. The ability to produce fragment could not be separated from the ability of alpha sarcin to inhibit protein synthesis. Alpha sarcin also causes the specific cleavage of the 23S RNA of the E. coli subunit to produce a smaller fragment of RNA than that produced from eukaryote ribosomes.

Specific in situ cleavage of 16S ribosomal RNA of Escherichia coli interferes with the function of initiation factor IF-1.

Specific in situ cleavage of 16S rRNA of E. coli has been accomplished by in vitro treatment of 70S ribosomes ("tight couples") with the bacteriocin cloacin DF13. The defective ribosomes, which have fully lost their ability to sustain polypeptide synthesis, are still able to form initiation on complexes with MS2 RNA, but the kinetics are altered. This is apparently due to an improper functioning of initiation factor IF-1, for the defective ribosomal couples respond normally to dissociation by IF-3 but the dissociation is not stimulated by IF-1. The initiation complexes formed with defective ribosomes are fully reactive with puromycin. Their ability to bind alanyl-tRNA is reduced by about 50% at all concentrations of elongation factor Tu studied. Cleavage of the 16S rRNA, not the release of the terminal fragment from the ribosome, causes the block of protein synthesis and the aberrations observed during initiation and elongation.

Requirement of protein synthesis for maturation of ribosomal RNA in Rhodopseudomonas spheroides

1. The role of protein synthesis in the maturation of ribosomal RNA was studied in the photosynthetic bacterium Rhodopseudomonas spheroides. 2. Methionine starvation and chloramphenicol both caused the accumulation of the 1.07 and 0.65 Mdalton ribosomal RNA precursors. 3. In methionine starved cultures accumulated precursor RNAs were processed upon addition of methionine. 4. The processing of these precursors upon methionine addition was inhibited in the presence of chloramphenicol. 5. It was concluded that protein synthesis is necessary for the cleavage of precursor ribosomal RNA.

HeLa cell nucleolar RNA synthesis after poliovirus infection

Infection of HeLa cell with poliovirus produces an early inhibition of host cell 45 S ribosomal RNA synthesis. The time of onset of inhibition is a function of the multiplicity of infection. Conversion of 45 S precursor RNA to 32 S ribosomal precursor RNA occurs at normal rates for some time after the onset of inhibition of synthesis and becomes depressed only at later times, suggesting that the effects of viral infection on cleavage and maturation of ribosomal RNA might be secondary to the decreased rate of formation of precursor. Prior to the onset of inhibition, there is a transient stimulation of ribosomal RNA synthesis. Infection in the presence of 3 m M guanidine causes a slower inhibition of RNA synthesis and enhances the detection of the stimulatory phase.

Colicin E3: a unique endoribonuclease.

The question of whether or not a cellular ribonuclease is involved in the cleavage of 16S ribosomal RNA by colicin E3 was investigated. For this purpose ribosomes from strains devoid of some ribonucleases or ribosomes in which ribonucleases had been inactivated by heat or removed by extensive washings were used for the colicin reaction. Since the 16S RNA of all these different ribosomes, and even of the most extensively washed ribosomes, was cleaved by colicin E3, it is suggested that cellular ribonucleases are not involved in colicin E3 action. Thus, colicin E3 seems to be a unique endoribonuclease.

The macromolecular metabolism of ribosomal precursor RNA as a common target of different antitumor drugs

The ability of mechloretamine, vincristine and daunomycin to interfere with methylation and cleavage of ribosomal precursor RNA has been tested in vitro in PHA-stimulated human lymphocytes. The cells were incubated with uridine- 3 H and 14 C-methyl-methionine for short periods, then the drugs were added to the medium and incubation continued for several hours. Lymphocyte RNA was extracted by a hot phenol method and analyzed on sucrose gradients. All the drugs studied caused a marked decrease in the number of radioactive methyl groups incorporated in ribosomal and transfer RNA. Accumulation of 3 H-uridine radioactivity was observed in 32S– 45S RNA. It is concluded that macromolecular metabolism of ribosomal precursor RNA is a common target of different antitumor drugs.

Effects of valine deprivation on ribosome formation in HeLa cells

When HeLa cells are deprived of the essential amino acid, valine, the formation of new ribosomes continues at a reduced rate compared to growing cells. Examination of nucleolar RNA reveals that during valine starvation there is a considerable decrease in the rate of 45 s ribosomal precursor RNA synthesis and of its conversion into 32 s RNA. Restoration of valine reverses these effects, but cycloheximide prevents reversal. The total steady-state level of nucleolar RNA remains practically constant for many hours after the onset of valine deprivation, but small changes in the relative amounts of 45 s and 32 s RNA occur. Preliminary experiments on cells deprived of other amino acids give results similar to those observed during valine deprivation. The findings indicate that in HeLa cells the availability of some particular protein or proteins may regulate the rate of synthesis and cleavage of ribosomal precursor RNA.