0022-3042/79/lWl-0931902 OOiO
Journul o f N u u r u c h ~ i i i t ~ I rVol y 33, pp. 931 10 937 Pergamon Press Ltd 1979. Printed in Great Britain 0 International Society lor Neurochemistry Ltd
RIBOSOMAL RNA PRECURSORS IN NEURONAL AND GLIAL RAT BRAIN NUCLEI A. S. STOYKOVA,’M. D. DABEVA,’ R. N. DIMOVA’ and A. A. HADJIOLOV’ ‘Regeneration Reseazch Laboratory and *Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria (Received 13 February 1979. Revised 6 April 1979. Accepted 11 April 1979)
Abstract-The method of THOMPSON (1973) for isolation and fractionation of brain nuclei was modified by the introduction of 12mM-Mg2+ in the isolating media. This technique gives a good yield of pure (85-90%) neuronal and glial rat brain nuclei, with minimal disruption of nuclei and degradation or processing of nuclear RNA. The RNA/DNA ratio of neuronal nuclei is about 3-fold higher than that of glial nuclei. Analysis of nucleolar RNA fractions by urea-agar gel electrophoresis allows the identification of 45S, 41s. 39S, 36S, 32s and 21s pre-rRNA components. The pattern of nucleolar pre-rRNA and rRNA species in neuronal and glial nuclei is identical. These results demonstrate the existence in brain nuclei of multiple pre-rRNA processing pathways qualitatively similar to those observed in other animal tissues.
THEPROCESSING of pre-rRNA plays an important role
pre-rRNA and rRNA in neuronal and glial nuclei is in the post-transcriptional regulatory mechanisms of identical, showing the simultaneous existence of mulribosome biogenesis in animal cells (HADJIOLOV& tiple pre-rRNA processing pathways in both types of NIKOLAEV, 1’976). Recently, the simultaneous existence brain nuclei. of multiple pre-rRNA processing pathways was MATERIALS AND METHODS & PENMAN, 1970), shown in HeLa cells (WEINBERG Labelling ofbruin R N A in vivo. Male Wistar albino rats BHK cell line 422E (WrNrKov, 1976), rat liver (HAD(15@180g) were lightly anaesthesized with diethyl ether JIOLOV et a!., 1974; DABEVA el a/., 1976; Dumv et al., 1978) and human lymphocytes (PURTELL & and given 25 pCi [14C]orotic acid (specific radioactivity ANTHONY,1975). The physiological role of the differ- 26 mCi/mmol) in a total volume of 50 p1 by intraventricuent pre-rRNA processing pathways has not yet been lar injection. The injection site (visualized with Toluidine Blue) was 2 mm to the right of the midsaggital hemisphere clarified. line, 2.5 mm posterior to the coronal suture and 4.5 mm The mechanism of ribosome formation in brain is deep from the cerebral skull surface. poorly understood due t o the difficulties inherent in Isolation of nuclei. All sucrose solutions contained studies with this organ. In experiments with whole 0.01 M-Tris-HC1 buffer, pH 6.5 and 1 or I2mM-MgCI2. brain or isolated brain nuclei from gold fish (CASOLA All manipulations were carried out at 4°C. The brains & AGRANOFF,1968) and rat (TENCHEVA & HADJIOLOV,(without the cerebellum and bulbi olfactorii) were rapidly 1969; SUNDE& SACHS,1972), chick embryos (JUDESet removed and placed in cold 0.14 M-NaCI. They were homal., 1971) or rat nucleoli (TAKAHASHI et a/., 1974) ogenized in 2.2~-sucrosewith 10 up and down strokes several distinct pre-rRNA species were identified. In of a loosely fitting glass-Teflon Potter-Elvehjem homogenthe only study with fractionated rat and rabbit brain izer. After dilution with the same solution the 1 5 2 0 % homogenate was filtered through two layers of cheesecloth. nuclei L0VTRUP-REIN (1970) found distinct differences laid over 1 ml 2.1 M-sucrose and centrifuged at 260,OOOy among the pre-rRNA species in neuronal, astrocytic for 30 min in the SW 40 rotor of a Beckman L5-50 ultraand oligodendroglial nuclei. centrifuge. The supernatant was aspirated, and the inside In this work we modified the method of THOMPSON of the tubes washed with 0.25 M-SUCTOSe and wiped thor(1973) for the isolation and fractionation of brain oughly. The resultant nuclear pellet in each tube was susnuclei with the aim of obtaining neuronal and glial pended in 3.5 ml of 2.3 M-sucrose. To each tube 1.5 ml of nuclei of high purity and minimal degradation of pre- 2.35 M-sucrose was underlaid and 2 ml of 1.8 M-sucrose and rRNA and rRNA. Analysis of RNA by urea-agar gel 6 ml of 0.25 M-SUCTOSe were overlaid and centrifuged at et al., 1976) showed the pres- 260,000 y for 30 min. The top 0.25 and 1.8 M-sucrose layers electrophoresis (DUDOV ence in total, neuronal and glial rat brain nuclei of were aspirated and discarded. The nuclei at the boundary between the 1.8 and 2.3 M-layers were collected, suspended all pre-rRNA species found previously in rat liver in 8 vol of 0.01 M-Tris-HC1 buffer (pH 6.5), containing (DABEVAef a/., 1976; 1978). The pattern of nucleolar 12 r n ~ - M g C and t ~ pelleted at 3000 y for 5 min. This pellet was designated ‘Pellet-”. The nuclear pellet from the bottom of the tubes was designated ‘Pellet-G’. Some experiAbbreuiation used: pre-rRNA : precursor to ribosomal ments involved two washes of the nuclear pellets with RNA. 0.25 M-sucrose. 93 1
932
A. S. STOYKOVA et al.
Light microscopy. The nuclear suspensions were fixed in
media with either 1 or 12 m - M g 2 + . Under the light formaldehyde for 10-15 min. Samples were taken, microscope pellet ' N isolated with 1 mM-Mg2+ constained with Toluidine Blue and differentiated in 96% eth- sisted almost entirely of large, lightly stained, finely anol. Nuclear counts were based on at least 200 nuclei, granulated nuclei with one or two distinct nucleoli. selected from random fields of the preparations. Numerous partly disrupted nuclei were seen. The'nuElectron microscopy. The nuclear pellets were fixed in clei from pellet 'N' isolated in the presence of 27; glutaraldehyde-0.1 M-cacodylatebuffer (pH 7.3-7.4) for 12 mwMg2' had similar characteristics, but under 2 h, followed by 2% OsO, for 1 h at 4°C. Ultrathin sections these conditions the nucleoplasm was roughly granuwere examined in a JEM-I00B electron microscope at lated. Partly disrupted nuclei were not seen. Electron 80 kV. Isolation of nuclear R N A . The extraction was carried microscopy (Fig. 1A) showed the nuclei to be free out at 50°C for I 5 min in a 1 : I (v/v) mixture of 0.1 M-Tris- of cytoplasm with well preserved nuclear membranes, acetate buffer (pH 5.5k1 mM-EDTA and phenol, saturated partly covered with ribosomes. The chromatin of with the same buffer and containing 0.1% 8-hydroxyquino- these nuclei was condensed in small clumps located line. The mixture was chilled on ice and centrifuged for mainly under the nuclear membrane and around the 20min at 30009 in the cold. The water phase was aspir- nucleolus. The chromatin of nuclei isolated in the ated and sodium dodecyl sulphate added to 0.5% followed presence of 1 mM-MgZ+ was dispersed. by an equal volume of phenol. After centrifugation the Light microscopy of pellet ' G showed that it conwater layer was aspirated, mixed with a 1 :1 (v/v) mixture of phenol-chloroform, shaken for 10 min and centrifuged sists of smaller and dark-stained nuclei. By electron as above. The last deproteinization of the water layer was microscopy the nuclei were free of cytoplasmic condonc with an equal volume of chloroform. R N A from the taminants, with preserved membranes (Fig. 1B). The final water phase was precipitated with 2 vol of 96% eth- chromatin was in large clumps which were more proanol, containing 1% potassium acetate at -10°C over- nounced in nuclei isolated in the presence of night. The RNA precipitate was collected by centrifugation 12 mM-Mg2+. Nucleoli were smaller and observed and washed 3 times with ethanol. The bulk of this RNA only in some nuclei. ef a/., 1978) and fraction is of nucleolar origin (DABEVA The condensation of chromatin caused by is designated as 5O"nu-RNA. In some experiments total 12mM-MgZ+in nuclei from both pellets 'N' and 'G' nuclear 50"RNA was obtained by the method of GEORGIEV was fully reversible when these nuclei were washed (1967). The 10% brain homogenate in 0.14~-NaCIwas twice with MgZ+-free 0.25 M-SUCrOSe, an observation mixed with an equal volume of phenol, saturated with (1973) with 0. I4 M-NaCI (pH 6) and containing 0.1% hydroxyquinoline. similar to that of LAVAL& BOUTEILLE The mixture was shaken for 15 min at 0°C and centrifuged liver nuclei. The nuclei from pellets ' N and 'G' were identified for 20 min at 3000 g. The water phase was discarded. The interphase layer was suspended in a 1:1 (v/v) mixture of on the basis of commonly used criteria (NURNBERGER, 0.14~-NaCI-phenol (as above) and shaken for 15min at 1964) showing that nuclei from pellet 'N are largely 50°C. The mixture, cooled on ice, was deproteinized 3-fold, neuronal, while pellet ' G contains mainly oligodenthe RNA precipitated and washed as above. This nuclear droglial and microglial nuclei. Contamination of RNA was designated 5O"RNA. The poly(A)'-RNA content neuronal nuclei by astroglial elements, constituting in the RNA fractions, determined by oligo(dT)-cellulose about 20% of brain cells (MORI & LEBLOND,1969) binding was below 3%. Urea-agar gel electrophoresis of R N A . This was carried has to be kept in mind. We did not attempt to evaluout as described by D u w v et al. (1976). The dried electro- ate the extent of such contamination since with isophoretograms were recorded at 260nm (TSANEV& lated nuclei it is practically impossible (MORI& LEB1967; OLPE et a!., STAYNOV, 1964). The labelled RNA components were LOND, 1970; KATO& KUROKAWA, detected by autoradiography and recorded at 550 nm 1974). Contamination of glial nuclei by granulated et a/., 1966). (TSANEV microneuronal nuclei (SMITHet al., 1976) should be Cheniical analyses. The amount of R N A and DNA was minimal in our case since the cerebellum and bulbi determined by the two-wave-length method (TSANEV & olfactorii were not included in the starting material. MAKKOV,1960). The purity of the nuclear fractions, estimated from Materials. Analytical grade reagents were used throughfive independent separations, was 88 1% for pellet out. Agar No. 1 was from Koch-Light Laboratories, Coln'N' and 85 f 1.8% for pellet 'G' (mean & s.D.). The brook, Bucks., U.K.; oligo(dT)cellulose was from Collaborative Research Inc., Waltham, MA, U.S.A. and yield of isolated neuronal plus glial nuclei, based on ['4C]orotate was from NAEC Institute for Isotopes, Buda- analyses of DNA content, was about 20%. Thus, cross-contamination between neuronal and glial nupest, Hungary. clei was only 12-15%, which permitted further studies of their biochemical characteristics. The RNA/DNA RESULTS AND DISCUSSION ratio (four experiments) for neuronal nuclei was The Mg2+ concentration currently used in the iso- 0.475 0.048, while it was only 0.155 5 0.020 for lation of brain nuclei (1-2 mM-Mg2+)results in appre- glial nuclei (mean s.D.). The RNA/DNA ratio for ciable disruption of neuronal nuclei and degradation total brain nuclei was 0.360 _+ 0.014. This ratio is of RNA. Therefore, we introduced 12 mM-Mg2+ into similar to previously reported values (HADJIOLOV et the isolation media. We carried out comparative mor- d.,1965; LOVTRUP-REIN & MCEWEN,1966; KATO& phological studies on pellets 'N' and 'G' isolated in KUROKAWA, 1967) and supplies independent evidence
4P,
FIG. I. Electron micrographs of the fractions of neuronal (A) and glial (B) rat brain nuclei isolated in the presence of 12 mM-MgZ+ followed by two washes in Mg2+-free 0.25 M-sucrose medium (see Materials and Methods).
933
935
Ribosomal RNA precursors in brain nuclei A 260
32 S
A
A550
10
0.6
0.6
OL
0.i
Distance from origin ( c m )
FIG. 2. Urea--agar gel electrophoresis of rat brain nuclear RNA. The brain nuclear R N A is labelled in uiuo with [14C]orotate for 2 h. A. Nucleolar RNA (50"nuRNA) extracted from isolated total brain nuclei. B. Nuclear (WRNA)extracted from the brain homogenate by subsequent treatment with phenol A s s o ; radioactivity recorded at 0 and 50°C (for details see Materials and Methods). ---, A 2 6 0 ; -,
from the radioautograms.
for their purity. A striking observation is that the 21s pre-rRNA, while the identity of the other two RNA/DNA ratio in neuronal nuclei is about 3 times remains to be established. The urea-agar gel electrophoresis profiles of nuhigher than in glial nuclei. These results are in agreeet a/., 1972) cleolar RNA extracted from isolated neuronal and ment with previous authors (AUSTOKER and indicate that neuronal nuclei are characterized glial nuclei were also investigated (Fig. 3). In both by a markedly higher RNA content of unknown types of brain nuclei the same pre-rRNA and rRNA species could be identified by their radioactivity and physiological significance. The electrophoretic profile of nucleolar 5O"nu-RNA (in some cases) , 4 2 6 0 profiles. The 45s. 36s and 32s obtained from isolated total brain nuclei is shown pre-rRNA predominated, while 415 and 21s prein Fig. 2A. As can be seen, nucleolar pre-rRNA and rRNA were less pronounced. The 39s component in rRNA constitute well delimited peaks appearing in nucleolar RNA from both neuronal and glial nuclei both the A 2 6 0 and radioactivity profiles. A similar was also clearly separated. These results show that pattern (Fig. 2B) was seen with nuclear RNA all six pre-rRNA components are typical of both (5O"RNA) prepared by direct homogenization of brain neuronal and glial nuclei. Therefore, the qualitative in phenol (see Materials and Methods), thus showing differences in the patterns of pre-rRNA species in that degradation and/or processing of pre-rRNA and neuronal, astroglial and oligodendroglial nuclei. rRNA during the isolation of nuclei is unlikely. In reported previously (L0VTRuP-REIN,1970) may be due both cases the peaks of 28s and 18s rRNA are well to the use of different fractionation techniques. delineated, providing additional evidence for the The simultaneous occurrence of 36S, 3 2 s and 2 IS 1967). pre-rRNA in brain nuclei demonstrates that multiple absence of degradation (VENKOV& HADJIOLOV, Five distinct RNA components moving slower than processing pathways operate in both neuronal and 28s rRNA can be identified. The three major peaks glial cells. In this respect, ribosome formation in brain co-migrate with the respective rat liver pre-rRNA and seems to follow the pattern established for other animal tissues (DABEVA et a/., 1976; DUDOV et d.,1978). can be identified as 45S, 36s and 32s pre-rRNA (DABEVA et al., 1976,1978). An RNA co-migrating The physiological role of the separate pre-rRNA prowith liver 41s pre-rRNA is seen as a minor radioac- cessing pathways has not yet been clarified. The pretivity peak. In contrast, the 39s pre-rRNA com- dominance of the 39s pre-rRNA component in neurer al., 1976) con- onal and glial nuclei is noteworthy. This component ponent, barely seen in liver (DABEVA stitutes a distinct fraction in brain. At least three may originate by a direct split of 28s rRNA from RNA peaks are found in the zone between 28s and 45s pre-rRNA (DABEVAer a/., 1976). The 28s rRNA 18s rRNA. One of these peaks coincided with liver generated in this way is likely to be functionally defi-
936
A. S. STOOYKOVA er a1
A 260 I
1.2
1.2
I
1.0
oe
0.6
0. L
0.2
0
1
2
3
L
5
6
0
Distance f r o m
1
2
3
4
5
6
origin ( c m 1
FIG.3. Urea-agar gel electrophoresis of nucleolar RNA (50"nuRNA) extracted from isolated rat brain A,,o; -, neuronal (A) and glial (B) nuclei. Labelling with ['4C]orotate in uiuo was for 2 h. A 5 5 0 ;radioactivity recorded from the radioautograms.
---.
cient since it does not contain 5.8s rRNA. Therefore, DLJDOVK. P.. DABEVAM. D., HADJIOLOV A. A. & the high levels of 39s Pre-rRNA in brain nuclei may TODOROV B. (1978) Processing and migration of riboindicate spurious processing of 45s p r e - r ~and ~ ~ nucleic acids in the nucleolus and nuc~eoplasmof rat liver 171, 375-383. of ,.RNA (cOOPER & G ~ ~ nuclei. ~ Biochem. ~ J. ~ , extensive G. P. (1967) The nature and biosynthesis of 1971). Detailed labelling kinetics studies are needed GEORCIEV nuclear ribonucleic acids. Prog. Nucl. Acid Res. Molec. in order to test this assumption. Biol. 6 , 259-351. HADJIOLOV A. A. & NIKOLAEV N. (1976) Maturation of ribosomal ribonucleic acids and the biogenesis of ribosomes. Progress Biophys. Molec. Biol. 31, 95-144. REFERENCES A. A., TENCHEVA Z. C. & BOJADJIEVA-MICHAIHADJIOLOV AUSTOKERJ., Cox D. & MATHIAS A. (1972) Fractionation LOVA A. G. (1965) Isolation and some characteristics of cell nuclei from brain cortex of adult cat. J . Cell. Biol. of nuclei from brain by zonal centrifugation and a study of RNA-polymerase activity in the various classes of nu26, 383-393. clei. Biochern. J . 129, 1139-1 155. HADJIOLOV A. A., DABEVA M. D. & M A C K E I ~ N S V.KV. I CASOLA L. & ACRANOFFB. W. (1968) Release of RNA from (1974) The action of a-amanitin in uiuo on the synthesis goldfish brain nuclei by sodium dodecyl sulfate. Biochem. and maturation of mouse liver ribonucleic acids. Biobiophys. Res. Commun. 30, 262-266. chem. J . 138, 321-334. COOPERH. & GIBSONE. (1971) Control of synthesis and JUDESC., JACOBM. & MANDEL P. (1971) Demonstration of the precursors of rRNA in brain cells by polyacrylwastage of ribosomal ribonucleic acid in lymphocy t es 4 I . The role of protein synthesis. J. biol. Chem., amide-gel electrophoresis. J. Neurochem. 18, 170-172. 246, 5059-5066. M. (1967) Isolation of cell nuclei KATOT. & KUROKAWA A. A,, EMANUI- from the mammalian cerebral cortex and their assortDABEVA M. D.. DLJDOVK. P., HADJIOLOV LOV I. & TODOROV B. (1976) Intranuclear maturation ment on a morphological basis. J. Cell Biol. 32, 649-662. pathways of rat liver ribosomal ribonucleic acids.' Bio- LAVALM. & BOUTEILLEM. (1973) Synthetic activity of chem. J. 160, 495-503. isolated rat liver nuclei--I. Ultrastructural study at A. A. & STOYK- various steps of isolation. E x p l Cell Res. 76, 337-348. DABEVA M. D., DLJDOV K. P., HADJIOLOV OVA A. S. (1978) Quantitative analysis of rat liver nucleo& MCEWENB. S. (1966) Isolation and fracLOVTRUP-REIN lar and nucleoplasmic ribosomal ribonucleic acids. Biotionation of rat brain nuclei. J . Cell Bid. 30, 405-415. chem. J . 171, 367-374. H. (1970) Synthesis of nuclear RNA in LOVTRLJP-REIN M. D. & HADJIOLOV A. A. (1976). DUDOVK. P., DABEVA nerve and glial cells. J . Neurochem. 17, 853-863. Simple agar/urea gel electrophoretic fractionation of MORIS. & LEBLONDC. P. (1969) Electron microscopic high molecular weight ribonucleic acids. Analyt. Biofeatures and proliferation of astrocytes in the corpus calchem. 76, 250-258. losum of the rat. J . romp. Neurol. 137, 197-226. Gwastage*
Ribosomal RNA precursors in brain nuclei
93 7
MORI S. & LtBLOm C. P. (1970) Electron microscopic TENCHEVA Z . & HADJIOLOV A. A. (1969) Characterization identification of three classes of oligodendrocytes and of rat brain ribonucleic acids by agar gel electrophoresis. J . Neurochem. 16, 769-776. a preliminary study of their proliferative activity in the R. J. (1973) Studies on RNA synthesis in two corpus callosum of young rat. J . comp. Neurol. 139, 1-30. THOMPSON J. I. (I964) In, Biology of Neurogliu (WINDLE, NURNBERCER populations of nuclei from the mammalian cerebral cortex. J . Neurochern. 21, 1 9 4 0 . ed.) p. 39. C. C. Thomas, Springfield, IL. OLPEH. R., HONECCERC. G., LEUBAG. & RABINOWICZ TSANEV R. G. & MARKOVG. G. (1960) Substances interferTH. (1974) The morphological characterization of isoing with spectrophotometric estimation of nucleic acids lated nuclei in comparison to in sitic nuclei from mouse and their elimination by the two wave length method. cortex. E x p l Bruiri Res. 21, 131-138. Biochim.-biophys. Actu 42, 442-452. PURTELL M. J. & ANTHONY D. D. (1975) Changes in ribo- TSANEV R. G. & STAYNOV D. Z. (1964) A method for direct somal RNA processing paths in resting and phytoultraviolet spectrophotometry of agar- and starch-gel electrophoregrams. Biokhimiyu 29, 1 126- I 13 I . hemagglu tinin-stimulated guinea-pig lymphocytes. Proc. TSANEV R. G., MARKOV G. G. & DFSSFVG. N. (1966) Innutn. Acad. Sci? U.S.A. 72. 3315-3319. P. J. & ZACON 1. S. (1976) SMITH S. J.. MCLAUCHLIN corporation of labelled precursors into the electroGranule neurons and their significance in preparation phoretic fractions of rat liver ribonucleic acid. Biochern. of isolated brain cell nuclei. Brairi Res. 103, 345J . 100, 204-210. 349. VENKOV P. V. & HADJIOLOV A. A. (1967) Characterization SUNIIED. & SACHSH. (1972) Fractionation of RNA of of rat liver nuclear RNA fractions obtained by thermal the rat neuronal lobe labelled in oifro: comparison with phenol fractionation. BiOchi/n. Bioph.v.7. .4ciu 142. RNA of cerebral cortex labelled in 11i1iu. Bruiri Res. 47, 276-279. 217-135. WEINBERG R. A. & P E N M A N S. (1970) Processing of 458 TAKAHASHI Y.. ARAM K . & IKEDA K . (1974) Isolation and nucleolar RNA. J . molec. Biol. 47, 16@17X. WINICOVI. (1976) Alternate temporal order in ribosomal some characteristics of nucleoli from rat brain. Bruiri Res. 73. 189-235. RNA maturation. J . molec. Biol. 100, I41 - 155.
N.C. 33/4--c
Journul o f N u u r u c h ~ i i i t ~ I rVol y 33, pp. 931 10 937 Pergamon Press Ltd 1979. Printed in Great Britain 0 International Society lor Neurochemistry Ltd
RIBOSOMAL RNA PRECURSORS IN NEURONAL AND GLIAL RAT BRAIN NUCLEI A. S. STOYKOVA,’M. D. DABEVA,’ R. N. DIMOVA’ and A. A. HADJIOLOV’ ‘Regeneration Reseazch Laboratory and *Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria (Received 13 February 1979. Revised 6 April 1979. Accepted 11 April 1979)
Abstract-The method of THOMPSON (1973) for isolation and fractionation of brain nuclei was modified by the introduction of 12mM-Mg2+ in the isolating media. This technique gives a good yield of pure (85-90%) neuronal and glial rat brain nuclei, with minimal disruption of nuclei and degradation or processing of nuclear RNA. The RNA/DNA ratio of neuronal nuclei is about 3-fold higher than that of glial nuclei. Analysis of nucleolar RNA fractions by urea-agar gel electrophoresis allows the identification of 45S, 41s. 39S, 36S, 32s and 21s pre-rRNA components. The pattern of nucleolar pre-rRNA and rRNA species in neuronal and glial nuclei is identical. These results demonstrate the existence in brain nuclei of multiple pre-rRNA processing pathways qualitatively similar to those observed in other animal tissues.
THEPROCESSING of pre-rRNA plays an important role
pre-rRNA and rRNA in neuronal and glial nuclei is in the post-transcriptional regulatory mechanisms of identical, showing the simultaneous existence of mulribosome biogenesis in animal cells (HADJIOLOV& tiple pre-rRNA processing pathways in both types of NIKOLAEV, 1’976). Recently, the simultaneous existence brain nuclei. of multiple pre-rRNA processing pathways was MATERIALS AND METHODS & PENMAN, 1970), shown in HeLa cells (WEINBERG Labelling ofbruin R N A in vivo. Male Wistar albino rats BHK cell line 422E (WrNrKov, 1976), rat liver (HAD(15@180g) were lightly anaesthesized with diethyl ether JIOLOV et a!., 1974; DABEVA el a/., 1976; Dumv et al., 1978) and human lymphocytes (PURTELL & and given 25 pCi [14C]orotic acid (specific radioactivity ANTHONY,1975). The physiological role of the differ- 26 mCi/mmol) in a total volume of 50 p1 by intraventricuent pre-rRNA processing pathways has not yet been lar injection. The injection site (visualized with Toluidine Blue) was 2 mm to the right of the midsaggital hemisphere clarified. line, 2.5 mm posterior to the coronal suture and 4.5 mm The mechanism of ribosome formation in brain is deep from the cerebral skull surface. poorly understood due t o the difficulties inherent in Isolation of nuclei. All sucrose solutions contained studies with this organ. In experiments with whole 0.01 M-Tris-HC1 buffer, pH 6.5 and 1 or I2mM-MgCI2. brain or isolated brain nuclei from gold fish (CASOLA All manipulations were carried out at 4°C. The brains & AGRANOFF,1968) and rat (TENCHEVA & HADJIOLOV,(without the cerebellum and bulbi olfactorii) were rapidly 1969; SUNDE& SACHS,1972), chick embryos (JUDESet removed and placed in cold 0.14 M-NaCI. They were homal., 1971) or rat nucleoli (TAKAHASHI et a/., 1974) ogenized in 2.2~-sucrosewith 10 up and down strokes several distinct pre-rRNA species were identified. In of a loosely fitting glass-Teflon Potter-Elvehjem homogenthe only study with fractionated rat and rabbit brain izer. After dilution with the same solution the 1 5 2 0 % homogenate was filtered through two layers of cheesecloth. nuclei L0VTRUP-REIN (1970) found distinct differences laid over 1 ml 2.1 M-sucrose and centrifuged at 260,OOOy among the pre-rRNA species in neuronal, astrocytic for 30 min in the SW 40 rotor of a Beckman L5-50 ultraand oligodendroglial nuclei. centrifuge. The supernatant was aspirated, and the inside In this work we modified the method of THOMPSON of the tubes washed with 0.25 M-SUCTOSe and wiped thor(1973) for the isolation and fractionation of brain oughly. The resultant nuclear pellet in each tube was susnuclei with the aim of obtaining neuronal and glial pended in 3.5 ml of 2.3 M-sucrose. To each tube 1.5 ml of nuclei of high purity and minimal degradation of pre- 2.35 M-sucrose was underlaid and 2 ml of 1.8 M-sucrose and rRNA and rRNA. Analysis of RNA by urea-agar gel 6 ml of 0.25 M-SUCTOSe were overlaid and centrifuged at et al., 1976) showed the pres- 260,000 y for 30 min. The top 0.25 and 1.8 M-sucrose layers electrophoresis (DUDOV ence in total, neuronal and glial rat brain nuclei of were aspirated and discarded. The nuclei at the boundary between the 1.8 and 2.3 M-layers were collected, suspended all pre-rRNA species found previously in rat liver in 8 vol of 0.01 M-Tris-HC1 buffer (pH 6.5), containing (DABEVAef a/., 1976; 1978). The pattern of nucleolar 12 r n ~ - M g C and t ~ pelleted at 3000 y for 5 min. This pellet was designated ‘Pellet-”. The nuclear pellet from the bottom of the tubes was designated ‘Pellet-G’. Some experiAbbreuiation used: pre-rRNA : precursor to ribosomal ments involved two washes of the nuclear pellets with RNA. 0.25 M-sucrose. 93 1
932
A. S. STOYKOVA et al.
Light microscopy. The nuclear suspensions were fixed in
media with either 1 or 12 m - M g 2 + . Under the light formaldehyde for 10-15 min. Samples were taken, microscope pellet ' N isolated with 1 mM-Mg2+ constained with Toluidine Blue and differentiated in 96% eth- sisted almost entirely of large, lightly stained, finely anol. Nuclear counts were based on at least 200 nuclei, granulated nuclei with one or two distinct nucleoli. selected from random fields of the preparations. Numerous partly disrupted nuclei were seen. The'nuElectron microscopy. The nuclear pellets were fixed in clei from pellet 'N' isolated in the presence of 27; glutaraldehyde-0.1 M-cacodylatebuffer (pH 7.3-7.4) for 12 mwMg2' had similar characteristics, but under 2 h, followed by 2% OsO, for 1 h at 4°C. Ultrathin sections these conditions the nucleoplasm was roughly granuwere examined in a JEM-I00B electron microscope at lated. Partly disrupted nuclei were not seen. Electron 80 kV. Isolation of nuclear R N A . The extraction was carried microscopy (Fig. 1A) showed the nuclei to be free out at 50°C for I 5 min in a 1 : I (v/v) mixture of 0.1 M-Tris- of cytoplasm with well preserved nuclear membranes, acetate buffer (pH 5.5k1 mM-EDTA and phenol, saturated partly covered with ribosomes. The chromatin of with the same buffer and containing 0.1% 8-hydroxyquino- these nuclei was condensed in small clumps located line. The mixture was chilled on ice and centrifuged for mainly under the nuclear membrane and around the 20min at 30009 in the cold. The water phase was aspir- nucleolus. The chromatin of nuclei isolated in the ated and sodium dodecyl sulphate added to 0.5% followed presence of 1 mM-MgZ+ was dispersed. by an equal volume of phenol. After centrifugation the Light microscopy of pellet ' G showed that it conwater layer was aspirated, mixed with a 1 :1 (v/v) mixture of phenol-chloroform, shaken for 10 min and centrifuged sists of smaller and dark-stained nuclei. By electron as above. The last deproteinization of the water layer was microscopy the nuclei were free of cytoplasmic condonc with an equal volume of chloroform. R N A from the taminants, with preserved membranes (Fig. 1B). The final water phase was precipitated with 2 vol of 96% eth- chromatin was in large clumps which were more proanol, containing 1% potassium acetate at -10°C over- nounced in nuclei isolated in the presence of night. The RNA precipitate was collected by centrifugation 12 mM-Mg2+. Nucleoli were smaller and observed and washed 3 times with ethanol. The bulk of this RNA only in some nuclei. ef a/., 1978) and fraction is of nucleolar origin (DABEVA The condensation of chromatin caused by is designated as 5O"nu-RNA. In some experiments total 12mM-MgZ+in nuclei from both pellets 'N' and 'G' nuclear 50"RNA was obtained by the method of GEORGIEV was fully reversible when these nuclei were washed (1967). The 10% brain homogenate in 0.14~-NaCIwas twice with MgZ+-free 0.25 M-SUCrOSe, an observation mixed with an equal volume of phenol, saturated with (1973) with 0. I4 M-NaCI (pH 6) and containing 0.1% hydroxyquinoline. similar to that of LAVAL& BOUTEILLE The mixture was shaken for 15 min at 0°C and centrifuged liver nuclei. The nuclei from pellets ' N and 'G' were identified for 20 min at 3000 g. The water phase was discarded. The interphase layer was suspended in a 1:1 (v/v) mixture of on the basis of commonly used criteria (NURNBERGER, 0.14~-NaCI-phenol (as above) and shaken for 15min at 1964) showing that nuclei from pellet 'N are largely 50°C. The mixture, cooled on ice, was deproteinized 3-fold, neuronal, while pellet ' G contains mainly oligodenthe RNA precipitated and washed as above. This nuclear droglial and microglial nuclei. Contamination of RNA was designated 5O"RNA. The poly(A)'-RNA content neuronal nuclei by astroglial elements, constituting in the RNA fractions, determined by oligo(dT)-cellulose about 20% of brain cells (MORI & LEBLOND,1969) binding was below 3%. Urea-agar gel electrophoresis of R N A . This was carried has to be kept in mind. We did not attempt to evaluout as described by D u w v et al. (1976). The dried electro- ate the extent of such contamination since with isophoretograms were recorded at 260nm (TSANEV& lated nuclei it is practically impossible (MORI& LEB1967; OLPE et a!., STAYNOV, 1964). The labelled RNA components were LOND, 1970; KATO& KUROKAWA, detected by autoradiography and recorded at 550 nm 1974). Contamination of glial nuclei by granulated et a/., 1966). (TSANEV microneuronal nuclei (SMITHet al., 1976) should be Cheniical analyses. The amount of R N A and DNA was minimal in our case since the cerebellum and bulbi determined by the two-wave-length method (TSANEV & olfactorii were not included in the starting material. MAKKOV,1960). The purity of the nuclear fractions, estimated from Materials. Analytical grade reagents were used throughfive independent separations, was 88 1% for pellet out. Agar No. 1 was from Koch-Light Laboratories, Coln'N' and 85 f 1.8% for pellet 'G' (mean & s.D.). The brook, Bucks., U.K.; oligo(dT)cellulose was from Collaborative Research Inc., Waltham, MA, U.S.A. and yield of isolated neuronal plus glial nuclei, based on ['4C]orotate was from NAEC Institute for Isotopes, Buda- analyses of DNA content, was about 20%. Thus, cross-contamination between neuronal and glial nupest, Hungary. clei was only 12-15%, which permitted further studies of their biochemical characteristics. The RNA/DNA RESULTS AND DISCUSSION ratio (four experiments) for neuronal nuclei was The Mg2+ concentration currently used in the iso- 0.475 0.048, while it was only 0.155 5 0.020 for lation of brain nuclei (1-2 mM-Mg2+)results in appre- glial nuclei (mean s.D.). The RNA/DNA ratio for ciable disruption of neuronal nuclei and degradation total brain nuclei was 0.360 _+ 0.014. This ratio is of RNA. Therefore, we introduced 12 mM-Mg2+ into similar to previously reported values (HADJIOLOV et the isolation media. We carried out comparative mor- d.,1965; LOVTRUP-REIN & MCEWEN,1966; KATO& phological studies on pellets 'N' and 'G' isolated in KUROKAWA, 1967) and supplies independent evidence
4P,
FIG. I. Electron micrographs of the fractions of neuronal (A) and glial (B) rat brain nuclei isolated in the presence of 12 mM-MgZ+ followed by two washes in Mg2+-free 0.25 M-sucrose medium (see Materials and Methods).
933
935
Ribosomal RNA precursors in brain nuclei A 260
32 S
A
A550
10
0.6
0.6
OL
0.i
Distance from origin ( c m )
FIG. 2. Urea--agar gel electrophoresis of rat brain nuclear RNA. The brain nuclear R N A is labelled in uiuo with [14C]orotate for 2 h. A. Nucleolar RNA (50"nuRNA) extracted from isolated total brain nuclei. B. Nuclear (WRNA)extracted from the brain homogenate by subsequent treatment with phenol A s s o ; radioactivity recorded at 0 and 50°C (for details see Materials and Methods). ---, A 2 6 0 ; -,
from the radioautograms.
for their purity. A striking observation is that the 21s pre-rRNA, while the identity of the other two RNA/DNA ratio in neuronal nuclei is about 3 times remains to be established. The urea-agar gel electrophoresis profiles of nuhigher than in glial nuclei. These results are in agreeet a/., 1972) cleolar RNA extracted from isolated neuronal and ment with previous authors (AUSTOKER and indicate that neuronal nuclei are characterized glial nuclei were also investigated (Fig. 3). In both by a markedly higher RNA content of unknown types of brain nuclei the same pre-rRNA and rRNA species could be identified by their radioactivity and physiological significance. The electrophoretic profile of nucleolar 5O"nu-RNA (in some cases) , 4 2 6 0 profiles. The 45s. 36s and 32s obtained from isolated total brain nuclei is shown pre-rRNA predominated, while 415 and 21s prein Fig. 2A. As can be seen, nucleolar pre-rRNA and rRNA were less pronounced. The 39s component in rRNA constitute well delimited peaks appearing in nucleolar RNA from both neuronal and glial nuclei both the A 2 6 0 and radioactivity profiles. A similar was also clearly separated. These results show that pattern (Fig. 2B) was seen with nuclear RNA all six pre-rRNA components are typical of both (5O"RNA) prepared by direct homogenization of brain neuronal and glial nuclei. Therefore, the qualitative in phenol (see Materials and Methods), thus showing differences in the patterns of pre-rRNA species in that degradation and/or processing of pre-rRNA and neuronal, astroglial and oligodendroglial nuclei. rRNA during the isolation of nuclei is unlikely. In reported previously (L0VTRuP-REIN,1970) may be due both cases the peaks of 28s and 18s rRNA are well to the use of different fractionation techniques. delineated, providing additional evidence for the The simultaneous occurrence of 36S, 3 2 s and 2 IS 1967). pre-rRNA in brain nuclei demonstrates that multiple absence of degradation (VENKOV& HADJIOLOV, Five distinct RNA components moving slower than processing pathways operate in both neuronal and 28s rRNA can be identified. The three major peaks glial cells. In this respect, ribosome formation in brain co-migrate with the respective rat liver pre-rRNA and seems to follow the pattern established for other animal tissues (DABEVA et a/., 1976; DUDOV et d.,1978). can be identified as 45S, 36s and 32s pre-rRNA (DABEVA et al., 1976,1978). An RNA co-migrating The physiological role of the separate pre-rRNA prowith liver 41s pre-rRNA is seen as a minor radioac- cessing pathways has not yet been clarified. The pretivity peak. In contrast, the 39s pre-rRNA com- dominance of the 39s pre-rRNA component in neurer al., 1976) con- onal and glial nuclei is noteworthy. This component ponent, barely seen in liver (DABEVA stitutes a distinct fraction in brain. At least three may originate by a direct split of 28s rRNA from RNA peaks are found in the zone between 28s and 45s pre-rRNA (DABEVAer a/., 1976). The 28s rRNA 18s rRNA. One of these peaks coincided with liver generated in this way is likely to be functionally defi-
936
A. S. STOOYKOVA er a1
A 260 I
1.2
1.2
I
1.0
oe
0.6
0. L
0.2
0
1
2
3
L
5
6
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Distance f r o m
1
2
3
4
5
6
origin ( c m 1
FIG.3. Urea-agar gel electrophoresis of nucleolar RNA (50"nuRNA) extracted from isolated rat brain A,,o; -, neuronal (A) and glial (B) nuclei. Labelling with ['4C]orotate in uiuo was for 2 h. A 5 5 0 ;radioactivity recorded from the radioautograms.
---.
cient since it does not contain 5.8s rRNA. Therefore, DLJDOVK. P.. DABEVAM. D., HADJIOLOV A. A. & the high levels of 39s Pre-rRNA in brain nuclei may TODOROV B. (1978) Processing and migration of riboindicate spurious processing of 45s p r e - r ~and ~ ~ nucleic acids in the nucleolus and nuc~eoplasmof rat liver 171, 375-383. of ,.RNA (cOOPER & G ~ ~ nuclei. ~ Biochem. ~ J. ~ , extensive G. P. (1967) The nature and biosynthesis of 1971). Detailed labelling kinetics studies are needed GEORCIEV nuclear ribonucleic acids. Prog. Nucl. Acid Res. Molec. in order to test this assumption. Biol. 6 , 259-351. HADJIOLOV A. A. & NIKOLAEV N. (1976) Maturation of ribosomal ribonucleic acids and the biogenesis of ribosomes. Progress Biophys. Molec. Biol. 31, 95-144. REFERENCES A. A., TENCHEVA Z. C. & BOJADJIEVA-MICHAIHADJIOLOV AUSTOKERJ., Cox D. & MATHIAS A. (1972) Fractionation LOVA A. G. (1965) Isolation and some characteristics of cell nuclei from brain cortex of adult cat. J . Cell. Biol. of nuclei from brain by zonal centrifugation and a study of RNA-polymerase activity in the various classes of nu26, 383-393. clei. Biochern. J . 129, 1139-1 155. HADJIOLOV A. A., DABEVA M. D. & M A C K E I ~ N S V.KV. I CASOLA L. & ACRANOFFB. W. (1968) Release of RNA from (1974) The action of a-amanitin in uiuo on the synthesis goldfish brain nuclei by sodium dodecyl sulfate. Biochem. and maturation of mouse liver ribonucleic acids. Biobiophys. Res. Commun. 30, 262-266. chem. J . 138, 321-334. COOPERH. & GIBSONE. (1971) Control of synthesis and JUDESC., JACOBM. & MANDEL P. (1971) Demonstration of the precursors of rRNA in brain cells by polyacrylwastage of ribosomal ribonucleic acid in lymphocy t es 4 I . The role of protein synthesis. J. biol. Chem., amide-gel electrophoresis. J. Neurochem. 18, 170-172. 246, 5059-5066. M. (1967) Isolation of cell nuclei KATOT. & KUROKAWA A. A,, EMANUI- from the mammalian cerebral cortex and their assortDABEVA M. D.. DLJDOVK. P., HADJIOLOV LOV I. & TODOROV B. (1976) Intranuclear maturation ment on a morphological basis. J. Cell Biol. 32, 649-662. pathways of rat liver ribosomal ribonucleic acids.' Bio- LAVALM. & BOUTEILLEM. (1973) Synthetic activity of chem. J. 160, 495-503. isolated rat liver nuclei--I. Ultrastructural study at A. A. & STOYK- various steps of isolation. E x p l Cell Res. 76, 337-348. DABEVA M. D., DLJDOV K. P., HADJIOLOV OVA A. S. (1978) Quantitative analysis of rat liver nucleo& MCEWENB. S. (1966) Isolation and fracLOVTRUP-REIN lar and nucleoplasmic ribosomal ribonucleic acids. Biotionation of rat brain nuclei. J . Cell Bid. 30, 405-415. chem. J . 171, 367-374. H. (1970) Synthesis of nuclear RNA in LOVTRLJP-REIN M. D. & HADJIOLOV A. A. (1976). DUDOVK. P., DABEVA nerve and glial cells. J . Neurochem. 17, 853-863. Simple agar/urea gel electrophoretic fractionation of MORIS. & LEBLONDC. P. (1969) Electron microscopic high molecular weight ribonucleic acids. Analyt. Biofeatures and proliferation of astrocytes in the corpus calchem. 76, 250-258. losum of the rat. J . romp. Neurol. 137, 197-226. Gwastage*
Ribosomal RNA precursors in brain nuclei
93 7
MORI S. & LtBLOm C. P. (1970) Electron microscopic TENCHEVA Z . & HADJIOLOV A. A. (1969) Characterization identification of three classes of oligodendrocytes and of rat brain ribonucleic acids by agar gel electrophoresis. J . Neurochem. 16, 769-776. a preliminary study of their proliferative activity in the R. J. (1973) Studies on RNA synthesis in two corpus callosum of young rat. J . comp. Neurol. 139, 1-30. THOMPSON J. I. (I964) In, Biology of Neurogliu (WINDLE, NURNBERCER populations of nuclei from the mammalian cerebral cortex. J . Neurochern. 21, 1 9 4 0 . ed.) p. 39. C. C. Thomas, Springfield, IL. OLPEH. R., HONECCERC. G., LEUBAG. & RABINOWICZ TSANEV R. G. & MARKOVG. G. (1960) Substances interferTH. (1974) The morphological characterization of isoing with spectrophotometric estimation of nucleic acids lated nuclei in comparison to in sitic nuclei from mouse and their elimination by the two wave length method. cortex. E x p l Bruiri Res. 21, 131-138. Biochim.-biophys. Actu 42, 442-452. PURTELL M. J. & ANTHONY D. D. (1975) Changes in ribo- TSANEV R. G. & STAYNOV D. Z. (1964) A method for direct somal RNA processing paths in resting and phytoultraviolet spectrophotometry of agar- and starch-gel electrophoregrams. Biokhimiyu 29, 1 126- I 13 I . hemagglu tinin-stimulated guinea-pig lymphocytes. Proc. TSANEV R. G., MARKOV G. G. & DFSSFVG. N. (1966) Innutn. Acad. Sci? U.S.A. 72. 3315-3319. P. J. & ZACON 1. S. (1976) SMITH S. J.. MCLAUCHLIN corporation of labelled precursors into the electroGranule neurons and their significance in preparation phoretic fractions of rat liver ribonucleic acid. Biochern. of isolated brain cell nuclei. Brairi Res. 103, 345J . 100, 204-210. 349. VENKOV P. V. & HADJIOLOV A. A. (1967) Characterization SUNIIED. & SACHSH. (1972) Fractionation of RNA of of rat liver nuclear RNA fractions obtained by thermal the rat neuronal lobe labelled in oifro: comparison with phenol fractionation. BiOchi/n. Bioph.v.7. .4ciu 142. RNA of cerebral cortex labelled in 11i1iu. Bruiri Res. 47, 276-279. 217-135. WEINBERG R. A. & P E N M A N S. (1970) Processing of 458 TAKAHASHI Y.. ARAM K . & IKEDA K . (1974) Isolation and nucleolar RNA. J . molec. Biol. 47, 16@17X. WINICOVI. (1976) Alternate temporal order in ribosomal some characteristics of nucleoli from rat brain. Bruiri Res. 73. 189-235. RNA maturation. J . molec. Biol. 100, I41 - 155.
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