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PHOSPHOLIPIDS AND NUCLEAR RNA
Cell Biology International, 1996, Vol. 20, No. 6, 407–412
PHOSPHOLIPIDS AND NUCLEAR RNA E. ALBI, M. MICHELI and M. P. VIOLA MAGNI Institute of General Pathology, University of Perugia, Policlinico Monteluce, 06100 Perugia, Italy Accepted 22 April 1996
It has been demonstrated that in hepatocyte nuclei the chromatin phospholipid fraction is localized near the RNA in decondensed chromatin. The aim of the present study was to see if there is any linkage between phospholipids and other nuclear components. Isolated hepatocyte nuclei and nuclear membranes were treated with deoxyribonuclease and ribonuclease. No loss of phospholipids was observed after DNA digestion, whereas 48% was lost following enzymatic RNA removal. This loss of phospholipids, localized either near the membrane or inside the nucleus, was not homogeneous for all phospholipids: phosphatidylserine and sphingomyelin being the most affected. It can be concluded that 48% of nuclear phospholipids, in particular sphingomyelin, is lost with RNA removal. This result is discussed in view of a possible role of ? 1996 Academic Press Limited phospholipids in DNA synthesis and RNA transcription. K: hepatocyte nuclei; nuclear membranes; RNA; phospholipids.
INTRODUCTION The presence of phospholipids (PLs) in chromatin has been demonstrated by histochemical and biochemical techniques in plant and animal cells (Chayen et al., 1959; Gahan, 1965; Viola Magni et al., 1985b). They seem to be associated with nucleoli (Rees et al., 1963), non-histone proteins (Manzoli et al., 1976) or the nuclear matrix (Cocco et al., 1980). Recently it has been shown that after lactoperoxidase radioiodination of isolated hepatocyte nuclei, chromatin PLs were unlabelled, whereas all the radioactivity was recovered in the nuclear membranes. This confirms that chromatin PLs are not due to contamination from the nuclear membrane PLs (Albi et al., 1994a). The chromatin PLs are 10% of those present in the entire nuclei and differ in composition (Viola Magni et al., 1985a; Albi et al., 1994a) and turnover (Viola Magni et al., 1986) from microsomal and nuclear PLs. During hepatic regeneration the chromatin PLs are synthesized in relation to DNA synthesis (Viola Magni et al., 1985a), and further changes are observed during hepatocyte maturation (Albi et al., 1991). In particular, the chromatin PLs contain a higher percentage of sphingomyelin (SP) enriched with polyunsaturated fatty acids than the nuclei 1065–6995/96/060407+06 $18.00/0
(Albi et al., 1994a). It is known that SP in vitro stimulates DNA-polymerase activity (Manzoli et al., 1981) and it has been demonstrated that there is an increased chromatin SP synthesis at the beginning of the S-phase in rat regenerating liver (unpublished data) and in rat hepatocytes during neonatal maturation (Albi et al., 1991). Changes in SP content have also been observed in different tumour cells (Splanger et al., 1975; Upreti et al., 1983). Concerning the nuclear localization of PLs, it has been demonstrated, by electron microscopy, using phospholipase A2 and ribonuclease (RNase) complexed with gold particles, that both RNA and PLs are present in decondensed chromatin near the nucleoli and nuclear membranes, in both hepatocytes (Fraschini et al., 1992) and erythroleukemic cells (Maraldi et al., 1992). These observations suggest a possible link between the RNA and PLs. To verify this possibility the nuclear PLs were analysed after enzymatic digestion of nuclei and nuclear membrane with RNase and deoxyribonuclease (DNase). The results show that DNase treatment does not cause any loss of PLs whereas RNase causes a loss of PLs from nuclei (48%) and from the nuclear membrane (33%). ? 1996 Academic Press Limited
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MATERIALS AND METHODS Materials DNaseI–RNase-free and RNase–DNase-free were obtained from Boehringer Mannheim (Milan, Italy); DNase I from Sigma (Sigma Chemical Co., St Louis, MO, U.S.A.). Animals Sprague–Dawley male and female rats, 60 days old, fed ad libitum and kept under a normal light–dark period were used for experiments. Hepatocyte nuclei preparations The hepatocyte nuclei were prepared according to Bresnick et al. (1967). Rats were anaesthetized and the livers were perfused via the portal vein with 0.25 sucrose+3.3 m CaCl2, homogenized in 2.2 sucrose+3.3 m CaCl2 and centrifuged at 100,000#g for 60 min. The pellet containing the nuclei was resuspended in 1 sucrose+1 m CaCl2 and centrifuged at 1500#g for 10 min. All solutions were adjusted to pH 7.4 with concentrated Tris. The nuclei were then washed twice with Barnes et al. (1957) solution. The entire procedure was carried out at 0–4)C. This method yields a homogeneous population of hepatocyte nuclei with no contamination from other types of nuclei present in the liver (Bresnick et al., 1967).
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Tris-ethylenediaminetetraacetic acid (EDTA) pH 7.7 (Davinson, 1972). For RNase digestion the Tris buffer was chosen instead of Barnes et al. (1957) solution, since the Mg2+ and Ca2+ present in this buffer inhibit enzyme activity (Eichorn et al., 1969; Morril and Reuss, 1969). EDTA was added to chelate the possible Mg2+ and Ca2+ ions which may remain after washings with Barnes et al. (1957) solution. The reaction mixtures contained nuclei equivalent to 14 mg protein, 5.5 mg DNA, 1.5 mg RNA, 0.6 mg PL and 150 ìg RNase–DNase-free (30 ìg/g of liver) in a final volume of 25 ml. Incubation was carried out by stirring at 37)C for 30 min and the reaction was stopped with an equal volume 10 m cold Tris-EDTA. The nuclei were centrifuged at 9000#g for 15 min and used for biochemical analysis. DNA-depleted nuclei preparation (DDN) The nuclei, washed in Barnes et al. (1957) solution, were resuspended in the same solution since the activity of DNase I is dependent on Mg2+ ions (Sambrok et al., 1989). The reaction mixtures contained nuclei equivalent to 14 mg protein, 5.5 mg DNA, 1.5 mg RNA, 0.6 mg PL and 1250 U DNase–I-RNase-free in a final volume of 25 ml. Incubation was carried out by stirring at 37)C for 20 min and the reaction was stopped with an equal volume of cold Barnes. The nuclei were centrifuged at 5000#g for 5 min and used for biochemical analysis. DDN RNase digestion
RNA isolation RNA was extracted from hepatocyte nuclei in three steps: (1) nuclei isolated in 2.2 sucrose; (2) nuclei isolated in 1 sucrose; and (3) nuclei isolated in Barnes et al. (1957) solution. The samples were mixed with 4 guanidium isothiocyanate, 5 m sodium citrate, 0.1 mercaptoethanol, 0.5% N-laurylsarcosine, pH 7.0 1:5 vol/vol (Chirgwing et al., 1979) and the RNA was isolated according to Sambrok et al. (1989). The isolated RNA had a ratio 260/280 between 1.7 and 2.0. The electrophoresis was carried out on 1% agarose/2.2 formaldehyde at 90 V for 1 h. Nuclei RNase digestion The nuclei were washed twice with Barnes et al. (1957) solution pH 7.2 and resuspended in 10 m
DDN in 10 m Tris-EDTA pH 7.7 were incubated with 150 ìg RNase–DNase-free at 37)C for 30 min, while stirring. The reaction was stopped with an equal volume of 10 m cold Tris-EDTA and the nuclei were centrifuged at 9000#g for 10 min. The double digestion was made to avoid the inhibitory action of DNA on RNase (Houck, 1957). Nuclear membrane isolation The nuclear membrane was separated from liver nuclei. Liver nuclei were isolated according to the procedure of Widnell and Tata (1964). Briefly, the tissue material was homogenized in 0.32 sucrose containing 3 m MgCl2, pH 7.4, and then centrifuged at 700#g for 10 min at 4)C. The pellet was
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resuspended in 2.4 sucrose containing 1 m MgCl2, pH 7.4 and the suspension centrifuged at 50,000#g for 60 min at 4)C. Nuclear membranes were isolated according to Kay and Johnston (1975). The isolated nuclei were lysed in 8 m Tris-HCl, pH 8.5, containing 0.1 m MgCl2, 4 m 2-mercaptoethanol (Merck, Darmstadt, Germany), 8% sucrose, 1 ìg/ml DNase I. After 15 min incubation at 22)C, the reaction was stopped by diluting the lysate with cold water (1:1, vol/vol). The lysed nuclei were centrifuged at 38,000#g for 15 min at 4)C, and the sediment resuspended in 10 m Tris HCl, pH 7.4, containing 0.1 m MgCl2, 5 m 2-mercaptoethanol, 10% sucrose, and 1 ìg/ml DNase I. The digestion was carried out for 20 min and stopped as described above. The final nuclear envelope pellet was obtained after centrifugation at 38,000#g for 15 min at 4)C. Nuclear membrane RNase digestion The reaction mixture contained the membranes equivalent to 2 mg protein, 40 ìg DNA, 40 ìg RNA, 0.5 mg PL and 150 ìg RNase–DNase-free (30 ìg/g of liver) in a final volume of 10 ml. The enzymatic digestion was made according to the procedure previously described.
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Table 1. Decrease in DNA, RNA and PLs content in hepatocite nuclei after enzymatic digestion
N N+RNAse DDN DDN+RNAse
DNA
RNA
PLs
404.4&42.8 396.8&28.7 29.1& 8.9* 20.5& 6.5*
116.5& 9.3 86.8&11.5† 102.8&15.6 27.8& 2.7‡
43.0&2.2 33.2&1.7§ 41.8&2.6 24.8&2.7§
Values are expressed as ìg/mg of proteins and indicated as means&SD of seven experiments for nuclei and three for enzymatic treatments. *P<0.005 vs DNA; †P<0.05 vs RNA; and ‡P<0.005 vs RNA; §P<0.005 vs PLs of nuclei. N=nuclei; DDN=depleted DNA nuclei.
once again. The second run consisted of chloroform:methanol:acetone:acetic acid:distilled water; 70:15:30:15:7.5 vol/vol. The lipids were detected with iodine vapour and scraped into test tubes for Pi determination.
RESULTS Hepatocyte nuclei composition
For each preparation DNA (Burton, 1956), RNA (Schneider, 1957), and protein (Lowry et al., 1951) contents were determined. The total amount of PL was determined by measuring the inorganic phosphorus present in both nuclei and membranes (Folch et al., 1975).
The nuclei contained a high concentration of DNA, about four times that of RNA, and a small amount of PL, approximately 1/10 of DNA (Table 1). The values expressed in ìg/mg of protein are on average higher than those reported in previous studies (Albi et al., 1994a), since in those experiments Phenyl Methylsulfonyl Fluoride (PMSF) was not used in the preparation and the proteases were not completely inhibited, hence a possible loss of protein cannot be excluded.
Biochemical determination of sphingomyelin
RNA analysis of hepatocyte nuclei
The SP content was determined by enzymatic assay using the method of Blaton et al. (1983).
The RNA of 2.2 sucrose nuclei showed the presence of ribosomal RNA bands (18S–28S). The ribosomal RNA decreased in 1 sucrose nuclei and was absent in Barnes et al. (1957) washed nuclei, thus showing that our nuclear preparations were not contaminated by endoplasmic reticulum (Fig. 1).
Biochemical determination
Lipid analysis The total lipids were extracted from each preparation with chloroform:methanol 2:1 vol/vol according to Folch et al. (1975) and the PLs were separated by TLC (thin layer chromatography) on silica gel in a bidimensional system using chloroform:methanol:ammonia, 65:25:4 vol/vol for the first run. The gels were dried and exposed to concentrated HCl vapour for 10 min, and dried
Nuclear composition in DNA-depleted nuclei The nuclei digested with DNase I contained nucleoli, RNA and ribonucleoproteins (Herman et al., 1978). The biochemical analysis of our
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80
%
60
40
20
0
Fig. 1. Electrophoresis of RNA from nuclei of hepatocytes: 1, Nuclei in 2.2 sucrose; 2, Nuclei in 1 sucrose; 3, Nuclei in Barnes et al. (1957) solution.
DDN preparation showed that DNA decreased by 97%, without any RNA and PLs loss (Table 1).
SP
PS
PI
PE
PC
Fig. 2. Differences in the loss of the individual PL after enzymatic digestion. The isolated hepatocyte nuclei were incubated with DNase I, RNase and DNase I+RNase and the PLs were extracted with methanol:chloroform (1:2) and chromatographed on TLC. After chromatography the single PLs were scraped and determined as Pi. No significant changes were observed in PI, PE and PC, whereas SP and PS decreased after DNase I+RNase treatment. The percentage of single PLs are indicated on the ordinate line and the PLs on the abscissa line. N=Nuclei, DDN=Depleted DNA Nuclei. ( =N; =N+RNase; =DDN; =DDN+RNase.)
8
PLs content in nuclei and in DDN after RNase digestion µg/mg proteins
6
After RNase digestion a 23% loss of PLs was observed together with a decrease of 26% of RNA. In DDN treated with RNase, the RNA decreased by 74% and also PLs content was reduced by 48%. The decrease in PLs was not homogeneous with regard to the single component. In non-treated nuclei phosphatidylcholine (PC) was 57.25%, phosphatidylethanolamine (PE) 23.10%, phosphatidylinositol (PI) 11.57%, phosphatidylserine (PS) 5.20%, SP 4.47% (Fig. 2) in agreement with previous results (Viola Magni et al., 1985b). In the nuclei treated with RNase there was an average decrease of 23% of the total PLs. RNase digestion in DDN doubled this loss. As regards the per cent distribution the SP decreased by 84% (from 4.47 to 0.78) and the PS showed a decrease of 45% (from 5.20 to 2.89), whereas the other PLs percentage remained practically unchanged with respect to that of isolated nuclei (Fig. 2). In order to confirm the chromatographic data, SP was determined separately using sphingomyelinase (Blaton et al., 1983). The results reported in Fig. 3 showed a decrease from 6.7&0.9 ìg/mg
4
2
0
N
DDN
N+ RNase
DDN+ RNase
Fig. 3. Quantitative changes of Sphingomyelin (ìg/mg protein) in hepatocyte nuclei after enzymatic digestion. N=Nuclei, DDN=Depleted DNA Nuclei. The isolated hepatocyte nuclei were incubated with DNase I, RNase, and DNase+RNase and the amount of SP was determined by an enzymatic reaction (Blaton et al., 1983). Nuclei+RNase: P<0.05 vs Nuclei; DDN+RNase: P<0.005 vs Nuclei. Values are expressed as ìg/mg of proteins and indicated as means of three experiments.
protein to 3.2&0.3 ìg/mg protein after double enzymatic digestion, thus confirming the loss of SP observed with the chromatographic method.
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Table 2. Decrease in DNA, RNA and PLs content in nuclear membranes after enzymatic treatment
M M+RNase
DNA
RNA
PLs
37.12&4.72 43.52&7.98
28.67&7.21 8.23&2.35*
253.93&27.14 172.07& 1.80†
Values are expressed as ìg/mg of proteins and indicated as means&SD of three experiments for control and three for enzymatic treatments. *P<0.05 vs RNA control and †P<0.005 vs PLs control. M=nuclear membrane.
Nuclear membrane The isolated nuclear membrane showed a strong reduction in nucleic acid, DNA was only 6% of that present in the nuclei (Table 2), so that the inhibitory action of DNA on RNA digestion is less relevant. After RNase digestion the RNA decreased by 72% and the PLs by 33% (Table 2), without significant changes in their composition. DISCUSSION The results of our investigation show that 48% of the PLs present in the nuclei are lost together with RNA after removal of DNA. The association of nucleic acids with membranes has been described in many biological models and it appears that this phenomenon plays a specific role. The sites of DNA anchorage represent the initial points for DNA synthesis. When the nascent DNA is labelled with a short pulse of H3-thymidine, the H3 DNA appears attached to a large, low density, possibly hydrophobic cellular component which has been interpreted as belonging to the nuclear membrane (Hildebrand and Tobey, 1973). The RNA which remains attached to the membrane is a large percentage of that present in the nuclei and its role is not completely clear. Active nuclear metabolism and RNA synthesis increase with pore frequency and the ribonuclear protein may stabilize the pore complex (Franke, 1974). The RNA linked to the membranes is relatively insensitive to RNase and is soluble in organic solvents (Leive, 1973). Previous results have shown the presence of PLs in isolated chromatin, characterized by a different composition and turnover with respect to those of nuclear membranes and microsomes (Albi et al., 1994a; Viola Magni et al., 1985a,b). Electron microscopy (EM) observations using RNase and phospholipase complexed with gold particles, have demonstrated that these PLs are localized in
uncondensed chromatin together with RNA (Fraschini et al., 1992). Treatment of nuclei with RNase causes a loss of PLs together with RNA. The loss is specific, since digestion of nuclei with DNase does not have any effect on the PLs. Nevertheless, the digested RNA is only 26% of the total RNA. This fraction is not ribosomal RNA possibly contaminating the nuclei, since we have shown by RNA electrophoresis that the nuclear preparations are ribosome-free. The limited action of RNase was attributed to the inhibitory effect of DNA on RNase action (Houck, 1957). The nuclei were therefore digested with DNase in order to remove the DNA. In this situation the RNA loss amounted to 74% of the total. However, 26% was still RNase resistant. The amount of PLs lost was 48%. Similar results were obtained on the preparation of nuclear membranes which contains only 6% of nuclear DNA, whereas almost the total RNA remains attached to the membranes. Digestion with RNase removed 72% of RNA and, also in this case, a considerable fraction of PLs. In both isolated nuclei and nuclear membrane RNA was lost together with PLs, thus confirming previous observation of a possible link between RNA and PLs (Fraschini et al., 1992). The analysis of PL composition did not show any difference in nuclear membrane before and after RNase digestion, thus showing a non-specific linkage with membrane PL. The same is not true for the nuclei in which the decrease in PLs is accompanied by a specific loss of SP and PS, thus supporting the hypothesis that the treatment of nuclei causes not only a loss of membrane PLs but also of another fraction of PLs which is part of chromatin structure as previously demonstrated (Albi et al., 1994a; Viola Magni et al., 1985a,b). In this case RNA is specifically linked to some PLs which seem to have a specific role. PS is a particular PL which can stimulate the activity of DNA-polymerase in vitro (Manzoli et al., 1981) and in vivo in hepatocytes during neonatal maturation, as it increases with the increased transcription for specific enzymes (Albi et al., 1991). The fact that 45% of PS is removed with RNase digestion strongly indicates that this link may have a functional significance, e.g. in the regulation of RNA synthesis or in nucleoprotein processing. SP has been shown to be a quantitatively major component of chromatin PLs (Albi et al., 1994a) and that it changes during cell duplication. In fact, in regenerating rat liver SP synthesis increases strongly at the beginning of S-phase (unpublished data). Variations of SP content have also been demonstrated in tumour cells (Splanger
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et al., 1975; Upreti et al., 1983) and in cells which have been induced to differentiate (Albi et al., 1994b). Concerning the possible link between RNA and PLs and its role, it may be suggested that RNA represents a possible anchorage for DNA to membrane and/or PLs. The greater efficiency of RNase on nuclei depleted of DNA can be taken to indicate that the RNA loses the DNA protection and the RNAse digestion removes the remnant of the complex. The presence of a complex PLs-RNA-DNA at the level of the nuclear membrane may represent the site for DNA synthesis (Franke, 1974). At the chromatin level the same complex may interact with specific PLs which could play a role in RNA transcription (Albi et al., 1991, 1994b). REFERENCES A E, M M, L C, T ML, V M MP, 1994a. Rat liver chromatin phospholipids. Lipids 29: 715–719. A E, S G, V M MP, 1994b. La sfingomielina nelle cellule eritroleucemiche di Freund. Atti XXII Congresso Naz.SIP Cagliari. p. 103. A E, V M MP, G PB, 1991. Chromatin phospholipids changes during rat liver development. Cell Biochem Funct 9: 119–123. B DWH, E MP, S LA, 1957. Some experiments in favour of the cellular hypothesis for the spleen curative factor. In De Hevey GC, Fersberg AG, Abbat JD, eds. Advances in Radiobiology. Oliver and Boyd. Edinburgh, 211–213. B V, D B M, S J, D B, 1983. Enzymatic assay for phosphatidylcholine and sphingomyelin in serum. Clin Chem 29: 806–809. B E, L K, S J, S A, Y DH, B H, H T, 1967. Isolation and ribonucleic acid synthesis in nuclei of rat fetal liver. Exp Cell Res 46: 396–411. B K, 1956. A study of the contaminations and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 62: 315–323. C J, G PB, L C LF, 1959. The masked lipids of nuclei. Quart J Microsc Sci 100: 279–284. C JM, P AE, MD RJ, R WJ, 1979. Isolation of biological ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294–5299. C L, M NM, M FA, 1980. Phospholipid interactions in rat liver nuclear matrix. Biochim Biophys Res Commun 96: 890–898. D JN, 1972. The Biochemistry of Nucleic Acid 7th Edn. Chapman and Hall. E GL, C P, E T, 1969. The interaction of metal ions with polynucleosides and related compounds J Biol Chem 244: 937. F WW, 1974. Structure, biochemistry, and functions of the nuclear envelope. Int Rev Cytol (Suppl. 4P): 71–236. F J, L M, S-S GH, 1975. A simple method for isolation and purification of total lipids from animal tissues. J Biol Chem 226: 497–509.
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F A, A E, G PB, V M MP, 1992. TEM cytochemical study of localization of phospholipids in interphase chromatin in rat hepatocytes. Histochemistry 97: 225–235. G PB, 1965. Histochemical evidence for the presence of lipids on the chromosomes of animal cells. Exp Cell Res 39: 136–144. H R, W L, P S, 1978. Heterogeneous nuclear RNA-protein fibers in chromatin-depleted nuclei. J Cell Biol 74: 241–250. H CE, T RA, 1973. Temporal organization of DNA in Chinese hamster cells: cell-cycle dependent association of DNA with membrane. Biochem Biophys Acta 331: 165. H JC, 1957. The inhibition of ribonuclease. Arch Biochem Biophys 26: 649–651. K RR, J IR, 1975. Rapid isolation of nuclear envelopes from rat liver. Meth Cell Biol 15: 277–284. L L, 1973. Bacterial Membranes and Walls. Dekker, New York. L OH, R NJ, F AL, R RJ, 1951. Protein measurement with the folin phenol reagent. J Biol Chem 193: 265–275. M FA, C S, M G, B O, M NM, 1981. Role of chromatin phospholipids on template availability and ultrastructure of isolated nuclei. Adv Enzyme Regul 20: 247–262. M FA, M JH, C S, B B, B S, 1976. Lipid-H1 nucleohistone interactions Mol Cell Biochem 10: 155–160. M NM, Z N, S S, D C R, S P, M FA, 1992. Intranuclear localization of phospholipids by ultrastructural cytochemistry. J Histochem Cytochem 40: 1383–1392. M GA, R MN, 1969. Inhibition of enzymatic degradation of RNA by bound calcium and magnesium. Biochim Biophys Acta 43: 179. R KR, R JH, V JS, 1963. The metabolism of isolated rat-liver nuclei and other subcellular fractions. Biochem J 86: 130–136. S J, F EF, M T, 1989. Molecular Cloning. A laboratory manual IIa edn. Cold Spring Harbor, New York Cold Spring Harbor Laboratory. S WC, 1957. Determination of nucleic acid in tissue by pentose analysis. Meth Enzymol 3: 680–684. S M, C ML, K SL, M HP, O P, 1975. Some biochemical characteristics of rat liver and Morris hepatoma nuclei and nuclear membranes. Cancer Res 35: 3131–3145. U GC, D A RJ, W R, 1983. Membrane lipids of hepatic tissue Phospholipids from subcellular fraction of liver and hepatoma J Nat Can Inst 70: 568–573. V M MP, G PB, A E, I R, G PF, 1985a. Synthesis of chromatin phospholipids and DNA synthesis in hepatic cells. Bas Appl Histochem 29: 253–259. V M MP, G PB, A E, I R, G PF, 1986. Phospholipids in chromatin: incorporation of [32P]2 -O in different subcellular fraction of hepatocytes. Cell Biochem Funct 4: 283–288. V M MP, G PB, P J, 1985b. Phospholipids in plant and animal cells Cell Biochem Funct 3: 71–78. W CC, T JR, 1964. A procedure for the isolation of enzymatically active rat-liver nuclei. Biochem J 92: 313–317.
PHOSPHOLIPIDS AND NUCLEAR RNA E. ALBI, M. MICHELI and M. P. VIOLA MAGNI Institute of General Pathology, University of Perugia, Policlinico Monteluce, 06100 Perugia, Italy Accepted 22 April 1996
It has been demonstrated that in hepatocyte nuclei the chromatin phospholipid fraction is localized near the RNA in decondensed chromatin. The aim of the present study was to see if there is any linkage between phospholipids and other nuclear components. Isolated hepatocyte nuclei and nuclear membranes were treated with deoxyribonuclease and ribonuclease. No loss of phospholipids was observed after DNA digestion, whereas 48% was lost following enzymatic RNA removal. This loss of phospholipids, localized either near the membrane or inside the nucleus, was not homogeneous for all phospholipids: phosphatidylserine and sphingomyelin being the most affected. It can be concluded that 48% of nuclear phospholipids, in particular sphingomyelin, is lost with RNA removal. This result is discussed in view of a possible role of ? 1996 Academic Press Limited phospholipids in DNA synthesis and RNA transcription. K: hepatocyte nuclei; nuclear membranes; RNA; phospholipids.
INTRODUCTION The presence of phospholipids (PLs) in chromatin has been demonstrated by histochemical and biochemical techniques in plant and animal cells (Chayen et al., 1959; Gahan, 1965; Viola Magni et al., 1985b). They seem to be associated with nucleoli (Rees et al., 1963), non-histone proteins (Manzoli et al., 1976) or the nuclear matrix (Cocco et al., 1980). Recently it has been shown that after lactoperoxidase radioiodination of isolated hepatocyte nuclei, chromatin PLs were unlabelled, whereas all the radioactivity was recovered in the nuclear membranes. This confirms that chromatin PLs are not due to contamination from the nuclear membrane PLs (Albi et al., 1994a). The chromatin PLs are 10% of those present in the entire nuclei and differ in composition (Viola Magni et al., 1985a; Albi et al., 1994a) and turnover (Viola Magni et al., 1986) from microsomal and nuclear PLs. During hepatic regeneration the chromatin PLs are synthesized in relation to DNA synthesis (Viola Magni et al., 1985a), and further changes are observed during hepatocyte maturation (Albi et al., 1991). In particular, the chromatin PLs contain a higher percentage of sphingomyelin (SP) enriched with polyunsaturated fatty acids than the nuclei 1065–6995/96/060407+06 $18.00/0
(Albi et al., 1994a). It is known that SP in vitro stimulates DNA-polymerase activity (Manzoli et al., 1981) and it has been demonstrated that there is an increased chromatin SP synthesis at the beginning of the S-phase in rat regenerating liver (unpublished data) and in rat hepatocytes during neonatal maturation (Albi et al., 1991). Changes in SP content have also been observed in different tumour cells (Splanger et al., 1975; Upreti et al., 1983). Concerning the nuclear localization of PLs, it has been demonstrated, by electron microscopy, using phospholipase A2 and ribonuclease (RNase) complexed with gold particles, that both RNA and PLs are present in decondensed chromatin near the nucleoli and nuclear membranes, in both hepatocytes (Fraschini et al., 1992) and erythroleukemic cells (Maraldi et al., 1992). These observations suggest a possible link between the RNA and PLs. To verify this possibility the nuclear PLs were analysed after enzymatic digestion of nuclei and nuclear membrane with RNase and deoxyribonuclease (DNase). The results show that DNase treatment does not cause any loss of PLs whereas RNase causes a loss of PLs from nuclei (48%) and from the nuclear membrane (33%). ? 1996 Academic Press Limited
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MATERIALS AND METHODS Materials DNaseI–RNase-free and RNase–DNase-free were obtained from Boehringer Mannheim (Milan, Italy); DNase I from Sigma (Sigma Chemical Co., St Louis, MO, U.S.A.). Animals Sprague–Dawley male and female rats, 60 days old, fed ad libitum and kept under a normal light–dark period were used for experiments. Hepatocyte nuclei preparations The hepatocyte nuclei were prepared according to Bresnick et al. (1967). Rats were anaesthetized and the livers were perfused via the portal vein with 0.25 sucrose+3.3 m CaCl2, homogenized in 2.2 sucrose+3.3 m CaCl2 and centrifuged at 100,000#g for 60 min. The pellet containing the nuclei was resuspended in 1 sucrose+1 m CaCl2 and centrifuged at 1500#g for 10 min. All solutions were adjusted to pH 7.4 with concentrated Tris. The nuclei were then washed twice with Barnes et al. (1957) solution. The entire procedure was carried out at 0–4)C. This method yields a homogeneous population of hepatocyte nuclei with no contamination from other types of nuclei present in the liver (Bresnick et al., 1967).
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Tris-ethylenediaminetetraacetic acid (EDTA) pH 7.7 (Davinson, 1972). For RNase digestion the Tris buffer was chosen instead of Barnes et al. (1957) solution, since the Mg2+ and Ca2+ present in this buffer inhibit enzyme activity (Eichorn et al., 1969; Morril and Reuss, 1969). EDTA was added to chelate the possible Mg2+ and Ca2+ ions which may remain after washings with Barnes et al. (1957) solution. The reaction mixtures contained nuclei equivalent to 14 mg protein, 5.5 mg DNA, 1.5 mg RNA, 0.6 mg PL and 150 ìg RNase–DNase-free (30 ìg/g of liver) in a final volume of 25 ml. Incubation was carried out by stirring at 37)C for 30 min and the reaction was stopped with an equal volume 10 m cold Tris-EDTA. The nuclei were centrifuged at 9000#g for 15 min and used for biochemical analysis. DNA-depleted nuclei preparation (DDN) The nuclei, washed in Barnes et al. (1957) solution, were resuspended in the same solution since the activity of DNase I is dependent on Mg2+ ions (Sambrok et al., 1989). The reaction mixtures contained nuclei equivalent to 14 mg protein, 5.5 mg DNA, 1.5 mg RNA, 0.6 mg PL and 1250 U DNase–I-RNase-free in a final volume of 25 ml. Incubation was carried out by stirring at 37)C for 20 min and the reaction was stopped with an equal volume of cold Barnes. The nuclei were centrifuged at 5000#g for 5 min and used for biochemical analysis. DDN RNase digestion
RNA isolation RNA was extracted from hepatocyte nuclei in three steps: (1) nuclei isolated in 2.2 sucrose; (2) nuclei isolated in 1 sucrose; and (3) nuclei isolated in Barnes et al. (1957) solution. The samples were mixed with 4 guanidium isothiocyanate, 5 m sodium citrate, 0.1 mercaptoethanol, 0.5% N-laurylsarcosine, pH 7.0 1:5 vol/vol (Chirgwing et al., 1979) and the RNA was isolated according to Sambrok et al. (1989). The isolated RNA had a ratio 260/280 between 1.7 and 2.0. The electrophoresis was carried out on 1% agarose/2.2 formaldehyde at 90 V for 1 h. Nuclei RNase digestion The nuclei were washed twice with Barnes et al. (1957) solution pH 7.2 and resuspended in 10 m
DDN in 10 m Tris-EDTA pH 7.7 were incubated with 150 ìg RNase–DNase-free at 37)C for 30 min, while stirring. The reaction was stopped with an equal volume of 10 m cold Tris-EDTA and the nuclei were centrifuged at 9000#g for 10 min. The double digestion was made to avoid the inhibitory action of DNA on RNase (Houck, 1957). Nuclear membrane isolation The nuclear membrane was separated from liver nuclei. Liver nuclei were isolated according to the procedure of Widnell and Tata (1964). Briefly, the tissue material was homogenized in 0.32 sucrose containing 3 m MgCl2, pH 7.4, and then centrifuged at 700#g for 10 min at 4)C. The pellet was
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resuspended in 2.4 sucrose containing 1 m MgCl2, pH 7.4 and the suspension centrifuged at 50,000#g for 60 min at 4)C. Nuclear membranes were isolated according to Kay and Johnston (1975). The isolated nuclei were lysed in 8 m Tris-HCl, pH 8.5, containing 0.1 m MgCl2, 4 m 2-mercaptoethanol (Merck, Darmstadt, Germany), 8% sucrose, 1 ìg/ml DNase I. After 15 min incubation at 22)C, the reaction was stopped by diluting the lysate with cold water (1:1, vol/vol). The lysed nuclei were centrifuged at 38,000#g for 15 min at 4)C, and the sediment resuspended in 10 m Tris HCl, pH 7.4, containing 0.1 m MgCl2, 5 m 2-mercaptoethanol, 10% sucrose, and 1 ìg/ml DNase I. The digestion was carried out for 20 min and stopped as described above. The final nuclear envelope pellet was obtained after centrifugation at 38,000#g for 15 min at 4)C. Nuclear membrane RNase digestion The reaction mixture contained the membranes equivalent to 2 mg protein, 40 ìg DNA, 40 ìg RNA, 0.5 mg PL and 150 ìg RNase–DNase-free (30 ìg/g of liver) in a final volume of 10 ml. The enzymatic digestion was made according to the procedure previously described.
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Table 1. Decrease in DNA, RNA and PLs content in hepatocite nuclei after enzymatic digestion
N N+RNAse DDN DDN+RNAse
DNA
RNA
PLs
404.4&42.8 396.8&28.7 29.1& 8.9* 20.5& 6.5*
116.5& 9.3 86.8&11.5† 102.8&15.6 27.8& 2.7‡
43.0&2.2 33.2&1.7§ 41.8&2.6 24.8&2.7§
Values are expressed as ìg/mg of proteins and indicated as means&SD of seven experiments for nuclei and three for enzymatic treatments. *P<0.005 vs DNA; †P<0.05 vs RNA; and ‡P<0.005 vs RNA; §P<0.005 vs PLs of nuclei. N=nuclei; DDN=depleted DNA nuclei.
once again. The second run consisted of chloroform:methanol:acetone:acetic acid:distilled water; 70:15:30:15:7.5 vol/vol. The lipids were detected with iodine vapour and scraped into test tubes for Pi determination.
RESULTS Hepatocyte nuclei composition
For each preparation DNA (Burton, 1956), RNA (Schneider, 1957), and protein (Lowry et al., 1951) contents were determined. The total amount of PL was determined by measuring the inorganic phosphorus present in both nuclei and membranes (Folch et al., 1975).
The nuclei contained a high concentration of DNA, about four times that of RNA, and a small amount of PL, approximately 1/10 of DNA (Table 1). The values expressed in ìg/mg of protein are on average higher than those reported in previous studies (Albi et al., 1994a), since in those experiments Phenyl Methylsulfonyl Fluoride (PMSF) was not used in the preparation and the proteases were not completely inhibited, hence a possible loss of protein cannot be excluded.
Biochemical determination of sphingomyelin
RNA analysis of hepatocyte nuclei
The SP content was determined by enzymatic assay using the method of Blaton et al. (1983).
The RNA of 2.2 sucrose nuclei showed the presence of ribosomal RNA bands (18S–28S). The ribosomal RNA decreased in 1 sucrose nuclei and was absent in Barnes et al. (1957) washed nuclei, thus showing that our nuclear preparations were not contaminated by endoplasmic reticulum (Fig. 1).
Biochemical determination
Lipid analysis The total lipids were extracted from each preparation with chloroform:methanol 2:1 vol/vol according to Folch et al. (1975) and the PLs were separated by TLC (thin layer chromatography) on silica gel in a bidimensional system using chloroform:methanol:ammonia, 65:25:4 vol/vol for the first run. The gels were dried and exposed to concentrated HCl vapour for 10 min, and dried
Nuclear composition in DNA-depleted nuclei The nuclei digested with DNase I contained nucleoli, RNA and ribonucleoproteins (Herman et al., 1978). The biochemical analysis of our
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80
%
60
40
20
0
Fig. 1. Electrophoresis of RNA from nuclei of hepatocytes: 1, Nuclei in 2.2 sucrose; 2, Nuclei in 1 sucrose; 3, Nuclei in Barnes et al. (1957) solution.
DDN preparation showed that DNA decreased by 97%, without any RNA and PLs loss (Table 1).
SP
PS
PI
PE
PC
Fig. 2. Differences in the loss of the individual PL after enzymatic digestion. The isolated hepatocyte nuclei were incubated with DNase I, RNase and DNase I+RNase and the PLs were extracted with methanol:chloroform (1:2) and chromatographed on TLC. After chromatography the single PLs were scraped and determined as Pi. No significant changes were observed in PI, PE and PC, whereas SP and PS decreased after DNase I+RNase treatment. The percentage of single PLs are indicated on the ordinate line and the PLs on the abscissa line. N=Nuclei, DDN=Depleted DNA Nuclei. ( =N; =N+RNase; =DDN; =DDN+RNase.)
8
PLs content in nuclei and in DDN after RNase digestion µg/mg proteins
6
After RNase digestion a 23% loss of PLs was observed together with a decrease of 26% of RNA. In DDN treated with RNase, the RNA decreased by 74% and also PLs content was reduced by 48%. The decrease in PLs was not homogeneous with regard to the single component. In non-treated nuclei phosphatidylcholine (PC) was 57.25%, phosphatidylethanolamine (PE) 23.10%, phosphatidylinositol (PI) 11.57%, phosphatidylserine (PS) 5.20%, SP 4.47% (Fig. 2) in agreement with previous results (Viola Magni et al., 1985b). In the nuclei treated with RNase there was an average decrease of 23% of the total PLs. RNase digestion in DDN doubled this loss. As regards the per cent distribution the SP decreased by 84% (from 4.47 to 0.78) and the PS showed a decrease of 45% (from 5.20 to 2.89), whereas the other PLs percentage remained practically unchanged with respect to that of isolated nuclei (Fig. 2). In order to confirm the chromatographic data, SP was determined separately using sphingomyelinase (Blaton et al., 1983). The results reported in Fig. 3 showed a decrease from 6.7&0.9 ìg/mg
4
2
0
N
DDN
N+ RNase
DDN+ RNase
Fig. 3. Quantitative changes of Sphingomyelin (ìg/mg protein) in hepatocyte nuclei after enzymatic digestion. N=Nuclei, DDN=Depleted DNA Nuclei. The isolated hepatocyte nuclei were incubated with DNase I, RNase, and DNase+RNase and the amount of SP was determined by an enzymatic reaction (Blaton et al., 1983). Nuclei+RNase: P<0.05 vs Nuclei; DDN+RNase: P<0.005 vs Nuclei. Values are expressed as ìg/mg of proteins and indicated as means of three experiments.
protein to 3.2&0.3 ìg/mg protein after double enzymatic digestion, thus confirming the loss of SP observed with the chromatographic method.
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Table 2. Decrease in DNA, RNA and PLs content in nuclear membranes after enzymatic treatment
M M+RNase
DNA
RNA
PLs
37.12&4.72 43.52&7.98
28.67&7.21 8.23&2.35*
253.93&27.14 172.07& 1.80†
Values are expressed as ìg/mg of proteins and indicated as means&SD of three experiments for control and three for enzymatic treatments. *P<0.05 vs RNA control and †P<0.005 vs PLs control. M=nuclear membrane.
Nuclear membrane The isolated nuclear membrane showed a strong reduction in nucleic acid, DNA was only 6% of that present in the nuclei (Table 2), so that the inhibitory action of DNA on RNA digestion is less relevant. After RNase digestion the RNA decreased by 72% and the PLs by 33% (Table 2), without significant changes in their composition. DISCUSSION The results of our investigation show that 48% of the PLs present in the nuclei are lost together with RNA after removal of DNA. The association of nucleic acids with membranes has been described in many biological models and it appears that this phenomenon plays a specific role. The sites of DNA anchorage represent the initial points for DNA synthesis. When the nascent DNA is labelled with a short pulse of H3-thymidine, the H3 DNA appears attached to a large, low density, possibly hydrophobic cellular component which has been interpreted as belonging to the nuclear membrane (Hildebrand and Tobey, 1973). The RNA which remains attached to the membrane is a large percentage of that present in the nuclei and its role is not completely clear. Active nuclear metabolism and RNA synthesis increase with pore frequency and the ribonuclear protein may stabilize the pore complex (Franke, 1974). The RNA linked to the membranes is relatively insensitive to RNase and is soluble in organic solvents (Leive, 1973). Previous results have shown the presence of PLs in isolated chromatin, characterized by a different composition and turnover with respect to those of nuclear membranes and microsomes (Albi et al., 1994a; Viola Magni et al., 1985a,b). Electron microscopy (EM) observations using RNase and phospholipase complexed with gold particles, have demonstrated that these PLs are localized in
uncondensed chromatin together with RNA (Fraschini et al., 1992). Treatment of nuclei with RNase causes a loss of PLs together with RNA. The loss is specific, since digestion of nuclei with DNase does not have any effect on the PLs. Nevertheless, the digested RNA is only 26% of the total RNA. This fraction is not ribosomal RNA possibly contaminating the nuclei, since we have shown by RNA electrophoresis that the nuclear preparations are ribosome-free. The limited action of RNase was attributed to the inhibitory effect of DNA on RNase action (Houck, 1957). The nuclei were therefore digested with DNase in order to remove the DNA. In this situation the RNA loss amounted to 74% of the total. However, 26% was still RNase resistant. The amount of PLs lost was 48%. Similar results were obtained on the preparation of nuclear membranes which contains only 6% of nuclear DNA, whereas almost the total RNA remains attached to the membranes. Digestion with RNase removed 72% of RNA and, also in this case, a considerable fraction of PLs. In both isolated nuclei and nuclear membrane RNA was lost together with PLs, thus confirming previous observation of a possible link between RNA and PLs (Fraschini et al., 1992). The analysis of PL composition did not show any difference in nuclear membrane before and after RNase digestion, thus showing a non-specific linkage with membrane PL. The same is not true for the nuclei in which the decrease in PLs is accompanied by a specific loss of SP and PS, thus supporting the hypothesis that the treatment of nuclei causes not only a loss of membrane PLs but also of another fraction of PLs which is part of chromatin structure as previously demonstrated (Albi et al., 1994a; Viola Magni et al., 1985a,b). In this case RNA is specifically linked to some PLs which seem to have a specific role. PS is a particular PL which can stimulate the activity of DNA-polymerase in vitro (Manzoli et al., 1981) and in vivo in hepatocytes during neonatal maturation, as it increases with the increased transcription for specific enzymes (Albi et al., 1991). The fact that 45% of PS is removed with RNase digestion strongly indicates that this link may have a functional significance, e.g. in the regulation of RNA synthesis or in nucleoprotein processing. SP has been shown to be a quantitatively major component of chromatin PLs (Albi et al., 1994a) and that it changes during cell duplication. In fact, in regenerating rat liver SP synthesis increases strongly at the beginning of S-phase (unpublished data). Variations of SP content have also been demonstrated in tumour cells (Splanger
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