Demethylation of ribosomal RNA by hepatocyte microsomal preparations

Life Sciences, Vol. 48, pp. Printed in the U.S.A.

1585-1589

Pergamon Press

D E M E T H Y L A T I O N OF RIBOSOMAL RNA BY HEPATOCYTE MICROSOMAL PREPARATIONS Won T. Choe, Christine L. Hatem, and Gary A. Clawson Department of Pathology, George Washington University Washington, DC 20037 (Received in final form February

12, 1991)

SUMMARY Previous work has shown that hypomethylation of rRNA is an important control for protein synthesis in rat hepatocytes, and that the net hypomethylation appears to arise from cytoplasmic events. Here we show that demethylation of rRNA is catalyzed by microsomal preparations. The rRNA demethylation is dependent upon NADPH and is almost completely inhibited by carbon monoxide. Demethylation appears to increase following galactosamine intoxication, a hepatotoxin which induces hypomethylation of rRNA and inhibition of protein synthesis.

A variety of hepatotoxins, including carbon tetrachloride (CCl4) , ethionine, and galactosamine (GAL), produce early defects in protein synthesis (1-5). These protein synthetic defects are all associated with profound defects in methylation of cytoplasmic rRNA (6-8): Protein synthesis shows a product-moment correlation coefficient of r = .95 with cytoplasmic rRNA methylation when a number of hepatotoxic settings are analyzed (7,8), whereas there is no significant correlation between nucleolar RNA m e t h y l a t i o n and protein synthesis (8). In contrast, early studies (generally employing rapidly-growing cultured cells) suggested that nearly all rRNA methylation occurred in the nucleolus, and that these methyl groups were conserved in mature cytoplasmic rRNA (9-12). Our data with quiescent hepatocytes do not seem compatiable with this assumption. For example, the specific activities of the cytoplasmic rRNA populations are considerably higher than those of nucleolar RNA, in spite of linear labeling conditions, the much larger pool size of the cytoplasmic rRNA, and the absence of a chase period (6,8). Additionally, with CCl 4 no defect in the nucleotide specificity of methylation of nucleolar RNA was observed, in spite of the fact that a specific 80% decrease in 2'-0-ribose methylations (specific for 28 and 18S rRNA) was found in cytoplasmic rRNA (6). These data demonstrate a turnover of methyl groups in mature cytoplasmic rRNA; such a cytoplasmic turnover would first require demethylation of methylated rRNA. Here, we localize demethylase activity to the microsomal fraction, and show that the d e m e t h y l a t i o n is inhibited by CO and is dependent upon NADPH. These results further substantiate the notion that demethylation of rRNA can occur in hepatocyte cytoplasm, and suggest that rRNA demethylation may be mediated by miorosomal cytochromes P450"

0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc

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METHODS Male, Sprague-Dawley rats were used with body weights of about 300 g. GAL was administered i.p. at a dose of 20 mg/100 g body weight, and rats were sacrificed at various intervals following treatment. Microsomal and S-IO0 fractions were obtained as described (6), and protein was quantitated by the method of Lowry et ai.3(13 ) . To obtain methylated cytoplasmic rRNA, rats were given 1.67 mCi of H-methyl-L-methionine (NEN, 70-85 Ci/mol) 2 h prior to sacrifice. Cytoplasmic RNA was isolated as described (6,14) using guanidine isothiocyanate/CsCl; recovery of tRNA is 0.1% using this method. Poly(A)+ RNA (which accounted for approximately 5% of the total methylation) was removed by oligo(dT)-cellulose chromatography, so that essentially all methylation was in 28 and 18S rRNA (6). Specific activities of these rRNA preparations was 7.7 ± 1.3 cpm/ug, and they were subsequently used as substrate for our demethylation studies in vitro. For demethylation studies, microsomal preparations (200 ug protein) were either untreated or exposed to CO for 60 sec by gently bubbling CO through the suspensions. As an anaerobic control, preparations were preexposed to N 2 in similar fashion. Assays contained i00 ug 3H-methylated rRNA, 200 ug microsomal preparations in TKM buffer (50 m M Tris-HCl, pH 7.5, 25 m M KCl, 5 mM MgC12) also containing 1 uM NADPH and i0 m M vanadyl ribonucleoside complexes. Demethylase activity was strictly dependent upon addition of fresh NADPH (see results). Incubations were for 60 min at 37 C. Samples included rRNA preparations incubated without microsomal preparations; microsomal preparations were added to these samples immediately prior to extraction. Following incubation, the mixtures were extracted with phenol, and aliquots were precipitated with ethanol overnight at 4 C. RNA was then recovered by centrifugation, and aliquots were assessed for radioactivity. Additional aliquots were taken for examination on 1.5% agarose gels (15), to insure that significant degradation did not occur. Other studies also examined the effects of imidazole (l,3-diaza-2,4cyclopentadiene; Sigma Chemical, #I0250) on rRNA demethylation. Imidazole is a nonspecific inhibitor which binds to cytochromes P450 with relatively high affinity and which inhibits a variety of P450-catalyzed reactions (16). RESULTS AND DISCUSSION In preliminary experiments, we utilized an in vitro assay to examine whether demethylation of rRNA could occur in the absence of significant rRNA degradation. Cytosolic (S-100) fractions did not exhibit demethylase activity, whereas microsomal preparations did. To further examine this phenomenon, we extended our investigation to include microsomal preparations taken at various periods following administration of GAL, an hepatotoxin which induces substantial decreases in protein synthesis and rRNA methylation (8). All microsomal preparations showed significant demethylation of cytoplasmic rRNA: Demethylase activity was slightly increased by 2 h following GAL administration, and was significantly increased by 4 h (Table i), where the extent of demethylation was greater than 30%. Since cytochrome P450 populations are an integral component of microsomes which possess a variety of demethylase activities (17,18), we examined whether the demethylation of 3H-methylated rRNA was affected by CO, using a somewhat unusual protocol, in which microsomal preparations were exposed to CO and subsequently incubated under aerobic conditions in

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demethylation reactions. Pre-exposure of microsomal p r e p a r a t i o n s to CO prior to aerobic incubations almost completely eliminated the demethylase activity; in all cases, the protection was statistically significant (Table i). A l t h o u g h unlikely, to control for the possibility that this resulted TABLE

I.

D e m e t h y l a t i o n of rRNA by Microsomal Preparations: Inhibition by Carbon Monoxide. Microsomal Source/ ± CO/ NADPH

Con/-CO/+NADPH Con/+CO/+NADPH Con/-CO/-NADPH GAL2/-CO/+NADPH GAL2/+CO/+NADPH GAL2/-CO/-NADPH GAL4/-CO/+NADPH GAL4/+CO/+NADPH GAL4/-CO/-NADPH Con Cytosol GAL4 Cytosol

NADPH D e p e n d e n c e and

Recovery of 3H-methylated rRNA (% control)

85 99 105 82 97 102 69 98 96 i00 94

± ! ± ~ ±

51'2 3 6 52 3

~ 142 ± 2

IValues represent means ± standard errors relative to values obtained with "late control" additions, from 3-10 separate experiments. GAL2 and GAL4 re present microsomal preparations obtained at 2 and 4 hours following administration of GAL. 2All -CO values (shown in bold type) differ from from the corresponding +CO values at p < 0.05 or greater, when compared using Student's t-test, and the GAL4/-CO demethylase activity differs significantly from the Con/-CO activity. No significant demethylase activity was observed in the absence of added NADPH, or with cytosol preparations.

from production of an anaerobic environment rather than from a COspecific effect, we also preexposed microsomal preparations to N 2. Preexposure to N 2 followed by aerobic incubations had no effect on the demethylation; demethylation following N 2 exposure was i00 ~ 5% and 97 ~ 8% in control and 2h GAL preparations (respectively). After storage, demethylase activity was strictly dependent upon addition of NADPH (Table I), a requirement which NADH did not fulfill. In additional studies, imidazole was added to standard rRNA demethylation reactions (at concentrations from 1-1000 uM) as a general inhibitor of cytochromes P450 reactions. We observed no inhibition of rRNA demethylation by imidazole, and in some experiments we observed a slight concentrat i o n - d e p e n d e n t increase in the extent of demethylation (not shown). Thus, the microsomal demethylation reaction may be catalyzed by h e m e - c o n t a i n i n g

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moieties other than cytochromes P450, or alternatively, they may be lyzed by specific isoforms not inhibited by imidazole (see below).

16, 1991

cata-

On the basis of our previous studies (including estimates based on minor changes in absorbance profiles) we estimated that perhaps i0 methyl groups/ribosome were involved in the hypomethylation (7), and in vitro m e t h y l a t i o n of ribosomes restored the protein synthetic capacity of the cytoplasmic populations. Furthermore, here about 15-30% of the 3H-methyl groups were removed during the incubations with microsomal preparations, which clearly indicates a global phenomenon rather than a peculiarity in appearance of radioactive label. Increases in the demethylase activity were associated with galactosamine treatment, and may at least in part underlie the cytoplasmic hypomethylation associated with this hepatotoxin. Such decreases, coupled with decreased methylase activity of S-100 fractions, may conspire to produce major changes in net methylation of cytoplasmic rRNA. Indeed, the hepatotoxins examined generally induce substantial decreases in S-adenosyl-methionine (and ATP) levels. In this regard, however, whereas ethionine treatment decreases ATP levels by about 90% in both males and females, males are relatively resistant to defects in protein synthesis and methylation of rRNA while females are not (7). Furthermore, males can be rendered susceptible to ethionine-induced changes in protein synthesis by pretreatment with estrogens (19), suggesting that a sex-dependent form of cytochrome P450 (P450 i) may be involved in the demethylation associated with this agent. Further, regulation of protein synthesis by rRNA methylation state may be peculiar to hepatocytes. Since cultured cells are routinely low in a variety of endoplasmic reticulum enzymes (including cytochromes P450)' it might be anticipated that significant net cytoplasmic hypomethylation would not be observed, since demethylase activity is a necessary prerequisite to the cytoplasmic turnover of methyl groups in rRNA. ACKNOWLEDGEMENTS We wish to thank Dr. Frank Gonzalez (NCI) for helpful discussion. This work was supported by a grant (2115R2) from the Council for Tobacco Research and an American Cancer Society summer fellowship to WTC. REFERENCES

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14. V. GLISIN, R. CRKVENJAKOV, and C. BYUS, Biochemistry 13, 2633-2637 (1974). 15. T. MANIATIS, E. FRITSCH, and J. SAMBROOK, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 204 (1983). 16. J. SHEETS, and J. MASON, Drug Metab. Dispos. 12, 603-606 (1984). 17. R. WHITE, and M. COON, Ann. Rev. Biochem. 49, 315-356 (1980). 18. D. NEBERT, and F. GONZALEZ, Ann. Rev. Biochem. 56, 945-995 (1987). 19. A. OLER, E. FARBER, and K. SHULL, Biochim. Biophys. Acta 190, 161-168 (1969).