319
Biochimica et Biophysica Acta, 390 (1975) 319--326 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98295
EFFECT OF THE EXOTOXIN OF BACILLUS THURINGIENSIS ON THE BIOSYNTHESIS AND MATURATION OF MOUSE LIVER NUCLEAR RNA
VLADIMIR V. MACKEDONSKI
Institute of Biochemistry, Bulgarian Academy of Sciences, 13 Sofia (Bulgaria) (Received November 26th, 1974)
Summary The effect of the exotoxin of Bacillus thuringiensis on the in vivo incorporation of [14 C] orotic acid into mouse liver nuclear rRNA and low molecular weight RNA was studied. The following results were obtained. 1. The exotoxin does not inhibit the synthesis of 45 S pre-rRNA, but causes a breakdown of these molecules. 2. The exotoxin inhibits the conversion of 38 S pre-rRNA into 32 S and 21 S. 3. The exotoxin inhibits the labelling of nuclear 5 S RNA, whereas the labelling of 4.6 S pre-tRNA is not affected. It is suggested that 5 S RNA may control the processing of 45 S prerRNA.
Introduction Previously we have shown that the exotoxin of Bacillus thuringiensis strongly inhibits the synthesis of nuclear rRNA species and the labelling of cytoplasmic 28 S, 18 S and 5 S rRNA in mouse liver, whereas the labelling of heterogeneous nuclear RNA and 4 S RNA is affected only to about 50% [1,2]. Recent investigations with the exotoxin on partially purified DNA dependent RNA polymerases have yielded controversal results [3,4]. However, our studies on the inhibition of RNA polymerase activities in isolated mouse liver nuclei by the exotoxin have suggested, that the in vivo action of this compound could not be explained by inhibition of RNA polymerases [5]. In this paper we report experiments on the synthesis and maturation of nuclear rRNA and nuclear lmwRNA in the presence of low doses of exotoxin. Abbreviations: pre-rRNA and pze-tRNA, precursors of r R N A and t R N A , respectively, ImwRNA, l o w m o l e c u l a r weight RNA.
320 The results show that the exotoxin does not inhibit the synthesis of 45 S pre-rRNA, but causes a random breakdown of a part of these molecules. On the other hand, the exotoxin interferes with the early steps of rRNA maturation. The synthesis of nuclear 4.6 S pre-tRNA is not affected, but the synthesis of nuclear 5 S RNA is inhibited. Part of these results have been reported [6]. Materials and Methods
Condition of labelling in vivo. The experiments were carried out with albino mice weighing 20--25 g. Highly purified B. thuringiensis exotoxin was dissolved in 0.14 M NaC1 and injected i.p. 30 min (or in some experiments 120 min) before the introduction of 25 pCi per mouse of [2 -14 C] orotic acid (spec. act. 18.5 Ci/mol). Isolation of nuclei. Highly purified nuclei were obtained by the method of Blobel and Potter [7], modified as described earlier [8]. Isolation and fractionation of nuclear RNA. Nuclear RNA was extracted from highly purified nuclei by phenol treatment at different temperatures [1,9] as follows: the nuclear pellet was suspended in ice cold 1 : 1 (v/v) mixture of 0.14 M NaC1 and phenol. The mixture was homogenized briefly, shaken at 4°C for 15 min and centrifuged at 4000 X g in the cold. RNA in the aqueous layer was further deproteinized with phenol, containing 0.5% sodium dodecylsulfate, phenol/chloroform (1 : 1 by vol.) and chloroform. RNA in the final aqueous layer was precipitated with 2.5 vol. of ethanol, containing 1% potassium acetate. This fraction was further used for preparation of nuclear lmwRNA by precipitating the high molecular weight RNA with 2 M NaC1 for 12 h at --10°C. The interphase layer obtained after 4°C extraction was suspended in 0.14 M NaC1/phenol mixture (1 : 1 by vol.) and shaken for 15 min at 50°C. RNA extracted in the aqueous layer was deproteinized as described above. This fraction contained the bulk of the nucleolar RNA components and was designated as nuclear rRNA. In pulse labelling experiments nuclear rRNA was isolated from a crude nuclear pellet prepared by low speed centrifugation of 10% liver homogenate in 0.25 M sucrose, containing 10 mM MgC12, 25 mM KC1 and 50 mM Tris • HC1 (pH 7.8). The pellet was treated twice with phenol]NaC1 mixture at 4°C and nuclear rRNA was extracted for 15 min at 50°C. This was done because during the preparation of highly purified nuclei some maturation of rRNA occurs (Mackedonski, V., unpublished observation). Gel electrophoresis of R N A fractions. Agar gel electrophoresis and radioautography were performed by the method of Tsanev and Staynov [10] under conditions described earlier [1]. Since a simple correlation exists between the sedimentation coefficient of RNA and its mobility in agar gel [11] the separate components were designated further in the text by their approximate S values. In some experiments the dried agar gel plates were cut into 1 mm slices and counted directly in PPO/POPOP/toluene scintillant in a model 3320 Packard liquid scintillation spectrometer. The lmwRNA was analyzed by polyacrylamide gel electrophoresis [12]. Horizontal gel slabs (18 cm × 10 cm) of 10% polyacrylamide cross-linked with 0.5% N,N'-methylenebisacrylamide were used. The buffer was 18 mM Tris •
321
HC1 (pH 7.8), containing 15 mM NaH2P04 and 0.5 mM EDTA. The gel was pre-run for 60 min and RNA fractionation was carried out at 80--100 V for 6 h at 20°C. The gel slabs were stained overnight with "stains all" (Eastman Organic Chem.) [13]. The gels were cut into 1 mm slices, put into counting vials, incubated for 24 h at room temperature and then for 1 h at 60°C in a mixture of toluene/water/Protosol® (10 : 1 : 9, by vol.), cooled and counted in PPO/POPOP/toluene scintillant with efficiency for ' 4C of about 70%. Determination of radioactivity. 1--2 A: 60 units of RNA were precipitated in 5% trichloracetic acid in the presence of 2 A260 units carrier tRNA in the cold for 20 min. The precipitate was filtered through Whatman GF-C filters and counted in PPO/POPOP/toluene scintillant. Reagents. Analytical grade reagents were used throughout. The phenol was freshly distilled, saturated with 0.14 M NaC1 and contained 0.1% 8-hydroxyquinoline. HighIy purified exotoxin of B. thuringiensis was kindly supplied by Dr K. Sebesta, Institute of Organic Chemistry and Biochemistry, CAS, Praha. 2-[ 14C]Orotic acid was obtained from NAEC Institute for Isotopes, Budapest. Results
Dependence o f nuclear rRNA labelling on the dose of exotoxin. Fig. 1 shows that the inhibitory effect of B. thuringiensis exotoxin on the in vivo labelling of nuclear rRNA depends on the dose. The labelling of nuclear rRNA is suppressed to about 60% of the control values by a dose of 0.025 mg per mouse. Higher doses (0.20 mg per mouse) completely inhibit the nuclear rRNA labelling. Labelling o f 45 S pre-rRNA in the presence of exotoxin. The synthesis of 45 S pre-rRNA was studied in pulse labelling experiments. When mice are injected i.p. with [14 C] orotic acid for 15 rain the label is incorporated mainly in the fraction of 45 S pre-rRNA (Fig. 2A). Several faint, but clear cut peaks with mobilities corresponding to 38 S, 32 S and 24 S are also seen on the radioautograms. 100 L.
U c
60
'5 >
40
u ~
20
0
\ &
d.2 d3 d.4
05
mg exotoxin per mouse
Fig. I . I n h i b i t i o n o f in vivo [ 1 4 C ] o r o t i c a c i d i n c o r p o r a t i o n i n t o n u c l e a r r R N A o f m o u s e liver b y t h e e x o t o x i n o f Bac. thuringiensis. T h e m i c e w e r e i n j e c t e d i.p. w i t h v a r y i n g a m o u n t s o f e x o t o x i n 3 0 m i n b e f o r e t h e a d m i n i s t r a t i o n o f t h e l a b e l l e d p r e c u r s o r . 9 0 m i n labelling t i m e .
322 325 285 1.2-
~;24s
10-
]J 118S
E
A
C
45Sll
g 06 ~ O4
y
0.2 0
2
4
6
0 2 4 Mobility, cm from stort
6
0
4
6
8
Fig. 2. Agar gel electrophoresis of m o u s e liver nuclear r R N A of control (A) and exotoxin treated (B and C) animals. Mice were pretreated with 0.025 m g exotoxin per m o u s e for 30 (B) and 1 2 0 (C) rain and after that 25 #Ci per m o u s e of [14C] orotic acid was injected i.p. 15 rain labelling time, - - , absorbance, recorded at 2 6 0 n m , - ..... , radioactivity, recorded f r o m the blackening of radioautogzams at 5 5 0 n m .
When mice are pretreated with exotoxin for 30 or 120 min [14 C] orotic acid incorporation into the total nuclear rRNA is not suppressed (Table I). However, the electrophoretic analyses, followed by radioautography shows (Figs 2B and 2C) that the distribution of the labelled RNA for exotoxin pretreated animals differs from that of the controls. The radioactivity in the 45 S pre-rRNA peak strongly decreases (see also Table I, last column), while certain amount of heterogeneous labelled RNA appears moving ahead of 45 S prerRNA. It should be noted, however, that 24 S RNA still persists as a sharp peak. It may be suggested, therefore, that the overall effect of the exotoxin is not an inhibition of the synthesis of 45 S pre-rRNA but its degradation to fragments with lower molecular weight. S t u d i e s on r R N A maturation. Further we studied whether 45 S pre-rRNA breaks down completely or it is processed into its normal products to a certain extent. Mice were pretreated with exotoxin for 30 min and [14 C] orotic acid was injected for 30 and 120 min. In this case the incorporation of [14 C] oro-
TABLE I EFFE CT OF B A C I L L U S T H U R I N G I E N S I S EXOTOXIN ON THE PULSE LA B ELLIN G OF MOUSE LI VER NUCL E AR rRNA* Extoxin pretreatm e n t (rain)
Labelling
Incorporation (% o f c o n t r o l )
(rain)
Nuclear rRNA ( c p m / A 2 6 0 nm u n i t RNA)
0 30 120
15 15 15
1030 1020 958
I00 100 93
time
45 S pre-rRNA peak** cpm
inc orp oration (% of control)
620 310 260
I00 50 42
* Exotoxin in a dose of 0.025 rag/mouse was injected i.p. 30 or 1 2 0 rain before the administration of 25 ~Ci/mouse of [14C] orotic acid. ** T h e radioactivity of 45 S p r e - r R N A w a s determined in the following way: the peak of 45 S w a s located after the radioautogzaphy of the electzophoregzams, cut out and counted cllzect~y in toluene/ P P O / P O P O P phosphor.
323 T A B L E II E F F E C T O F B A C I L L U S T H U R I N G I E N S I S E X O T O X I N ON THE [ 1 4 C ] O R O T I C A C I D I N C O R P O R A T I O N IN M O U S E L I V E R N U C L E A R r R N A E x o t o x i n w a s administered i.p. 3 0 m i n b e f o r e [ 14C] orotic acid. Labelling time (min)
Radioactivity ( c p m / A 2 6 0 n m unit R N A ) Con~ol
Exotoxintreated
9430 13 5 3 5
5450 8987
30 120
Incorporation (% o f c o n t r o l )
57.8 66.4
rate into the total nuclear rRNA was inhibited to 60--65% of the controls (Table II). Nuclear rRNA was isolated from control and exotoxin-treated animals and was analyzed by agar gel electrophoresis. The A260nm profile of the nuclear rRNA shows the presence of 45 S, 32 S, 28 S, 21 S and 18 S peaks comparatively well separated (Figs 3A and 3B). Exotoxin pretreatment produces characteristic changes in the A260nm profile of nuclear rRNA (Figs 3C and 3D). The relative amounts of 45 S, 32 S and 21 S pre-rRNA peaks progressively decrease, compared to those of 28 S and 18 S rRNA peaks. Similar changes are observed with the newly synthesized RNA after both 30 and 120 min labelling. In controls the newly synthesized rRNA includes 45 S pre-rRNA and its normal maturation products, 32 S, 24 S and 21 S, after 30 min labelling and 32 S, 28 S, 21 S, 18 S and 15 S after 120 min labelling. 1.0
32S 28S
A
B
O.8 ~21S Q6
/,
0.4 E Q2 c O
0 L 1.0
C
D
0
fi = 0.8
0
"~ Q6 0.4 02 2
4 6 0 2 4 Mobility, cm from start
6
8
Fig. 3. Agar gel electxophoresis o f m o u s e liver nuclear r R N A o f c o n t r o l ( A and B) and e x o t o x i n treated (C and D) intmal~. Mice w e r e pretreated w i t h 0 . 0 2 5 m g e x o t o x i n per m o u s e and 3 0 m i n later [ 14C] o r o t i c acid w a s injected for 3 0 (A and C) and 1 2 0 (B and D) rain. - - , absorbance, r e c o r d e d at 2 6 0 n m , ...... , radioactivity, r e c o r d e d from the b l a c k e n i n g o f r a d i o a u t o g z a m s at 5 5 0 n m .
324
Q8 07 0.6 0.5 0.4 Q3 Q2 E c Q1 o
5S
o
~: 0.7 06 0.5 O4
4.6S
4S A
~20
c
!'
i',,
4o
E c
050 8
C
'200 '> '150
o.3!
100
0.2 I 01:
SO
.o "iJ
u
0
0
Direction of
run
Fig. 4. A c r y l a m i d e gel e l e c t r o p h o r e s i s o f m o u s e liver n u c l e a r I m w R N A f r o m c o n t r o l ( A and C) and e x o t o x i n t r e a t e d (B and D ) animals. Mice w e r e i n j e c t e d w i t h 0 . 0 2 5 m g e x o t o x i n per m o u s e and 3 0 m i n later [ 1 4 C ] o r o t i c acid w a s given i.p. for 3 0 ( A and B) and 1 2 0 (C and D ) min. - . . . . . , c h r o m o s c a n , r e c o r d e d at 5 5 0 n m ; - - - , radioactivity.
The same R N A species are presented also after pretreatment with exotoxin. However, characteristic changes are observed. The amount of radioactivity in 45 S, 32 S and 21 S rRNA peaks is markedly decreased as compared to that in 28 S and 18 S rRNA peaks. The labelling of 24 S R N A peak, however, is less (or is not) affected, as in the case of shorter labelling time (see Fig. 2). The results reveal that the 45 S pre-rRNA molecules synthesized in the presence of e x o t o x i n may undergo further processing, but at a slower rate, especially in the case of 38 S -* 32 S and 21 S conversion, as discussed below. Labelling of 5 S, 4.6 S and 4 S RNA. We have previously shown that higher doses of e x o t o x i n inhibited the labelling of 5 S rKNA almost completely, while the labelling of 4 S t R N A was less affected [ 1 , 6 ] . Here we confirm our observations with lower doses of exotoxin. We have found, that 5 S and 4.6 S RNA, the latter being identified as a precursor of t R N A [14,15] are more highly labelled than 4 S R N A is (Figs 4A and 4C). After treatment with e x o t o x i n for 30 (Figs 4A and 4B) or 120 min (Figs 4C and 4D) the specific radioactivity of 5 S R N A is about 50% of the control, while the labelling of 4.6 S R N A is unaltered. The labelling of 4 S R N A is only slightly affected (about 90% of the control). It should be noted that after 30 and 120 min labelling the incorporation of 14 C into 5 S R N A is inhibited to the same extent by the e x o t o x i n as that of 45 S pre-rRNA (see Fig. 2). Similarly, in our early experiments with higher doses of exotoxin, the labelling of 28 S and 18 S rRNA was found to be suppressed to the same degree as was the labelling of 5 S rRNA [1,6]. Discussion Our results have shown that the e x o t o x i n does not inhibit the synthesis of 45 S pre-rRNA but causes a rapid breakdown of a part of these molecules. The
325
45 S pre-rRNA molecules are synthesized in 2--5 min and have an average life time of about 15 min [16,17]. Consequently, in our pulse labelling experiments the radioactivity should be incorporated mostly into 45 S pre-rRNA. Since the low doses of exotoxin used in these experiments do not suppress the labelling of RNA (Table I) one could assume that the exotoxin does not inhibit the synthesis of 45 S pre-rRNA. On the other hand, the radioautograms show that in exotoxin treated animals 45 S pre-rRNA is much less labelled, while a heterogeneous population of RNA molecules with lower average molecular weight appears, indicating that most probably the exotoxin causes a rapid and. random degradation of a part of the 45 S pre-rRNA molecules. Another possibility is that the exotoxin removes the unterminated rRNA fragments from the DNA template, but it is less likely, because of the following reasons: 24 S RNA is believed to be the nonconservative part of pre-rRNA, located at the 3' end of the 45 S pre-rRNA molecule [18,19]. In the first step of rRNA processing 45 S pre-rRNA is split by an exonucleolytic scission into 38 S and 24 S RNA [20,21] (see the scheme). The presence of 24 S RNA peak even after 120 min exotoxin pretreatment strongly suggests that the exotoxin does not impede the synthesis of 45 S pre-rRNA. ~24S 45 S I)
I exotoxin
~ 32 S
~38S
, 28 S
lexoox
After exotoxin treatment the labelling of 32 S and 21 S pre-rRNA equals t h a t of 28 S and 18 S rRNA, respectively. This phenomenon could result either from an increase in the rate of 32 S and 21 S pre-rRNA maturation, or from a decrease in the rate of their formation. The first possibility is unlikely because, as we have previously shown, the labelling of cytoplasmic 28 S and 18 S rRNA species is suppressed to the same extent as is the labelling of the nuclear rRNA [1]. On the other hand, it is possible that the exotoxin inhibits the conversion of 45 S pre-rRNA into 38 S and 24 S. However, at 15 and 30 min labelling times (Figs 2 and 3C) the labelling of 24 S RNA is less (or is not) affected by the exotoxin as compared to that Of 32 S and 21 S pre-rRNA. Since 45 S pre-rRNA cleavage gives 24 S RNA and 38 S pre-rRNA, the latter being a common precursor of both 32 S and 21 S pre-rRNA (see the scheme), then these results indicate that the exotoxin interferes with the conversion of 38 S into 32 S and 21 S pre-rRNA. Studies on the nuclear lmwRNA have shown that 5 S and 4.6 S RNA have different sensitivity to the exotoxin. Since our results with alpha-amanitin [8] have shown that 5 S RNA is inhibited, while 4.6 S RNA is not, it may be suggested that 5 S and 4.6 S RNA are synthesized by different mechanisms (or polymerases). Finally, we should like to point out that the synthesis of nuclear 5 S RNA is inhibited to the same extent as is the labelling of 45 S pre-rRNA at both 30 and 120 min labelling. A similar correlation we observed with higher doses of exotoxin: the labelling of 28 S, 18 S and 5 S rRNA was inhibited to about 90%
326 (see ref. 1). Since the synthesis of 5 S RNA is not affected in the absence of rRNA synthesis and ribosome assembly [23,24] and since it is known that 5 S RNA is integrated into 80 S (or 55 S) ribosomal precursor ribonucleoprotein in the nucleolus [22], our results suggest, but do not prove, the possibility that 5 S RNA could control the turnover of pre-rRNA in the nucleolus. The normal processing of 80 S ribosomal precursor ribonucleoproteins in the nucleolus might depend on their proper assembly, so that the particles lacking 5 S RNA might be more susceptible to degradation. Acknowledgements I wish to thank Professor A.A. Hadjiolov for the helpful discussion, G.N. Dessev for the help with the manuscript and D. Kulekova for her excellent technical assistance. References 1 Mackedonski, V.V., Nikolaev, N., Sebesta, K. and Hadjiolov, A.A. (1972) Biochim. Biophys. Acta 272, 56--66 2 Maekedonski, V.V., Hadjiolov, A.A. and Sebesta, K. (1972) FEBS Lett. 21, 211--214 3 Smuckler, E.A. and Hadjiolov, A.A. (1972) Biochem. J. 129, 153--166 4 Beebee, T., Korner, A. and Bond, R.P. (1972) Bioehem. J. 127, 619--624 5 Maekedonski, V.V. and Hadjiolov, A.A. (1974) Dokl. Bolg. Acad. Nauk. 27, 1117--1120 6 Mackedonski, V.V. (1973) Ph.D. Thesis, Institute of Biochemistry, Sofia 7 Blobel, G. and Potter, V.R. (1966) Science 154, 1662--1665 8 Hadjiolov, A.A., Dabeva, M.D. and Mackedonski, V.V. (1974) Biochem, J. 138, 321--334 9 Markov, G.G. and Arion, V.J. (1973) Eur. J. Biochem. 33, 186--200 10 Tsanev, R.G. and Staynov, D.Z. (1964) Biokhimyia 27, 1126--1131 11 Hadjiolov, A.A., Venkov, P.V. and Tsanev, R.G. (1966) Anal. Biochem. 17, 263--267 12 Loening, U.E. (1970) Method Med. Res. 12, 359--368 13 Dahalberg, A.E.0 Dingman, C.W. and Peacock, A.C. (1969) J. Mol. Biol. 41, 139--152 14 Burdon, R.H. (1971) Progr. NucL Acid Res. Mol. Biol. 11, 33--79 15 Choe, B.K. and Taylor, M.N. (1972) Biochim. Biophys. Acta 272, 275--287 16 Chaudhuri, S. and Lieberman, I. (1968) J. Mol. Biol. 33, 323--326 17 Greenberg, H. and Penman, S. (1968) J, Mol. BioL 21, 527--536 18 Wallauer, P.K. and David, I.B. (1973) Proc. Natl. Acad. Sei. U.S. 70, 2827--2831 19 Hadjiolov, A.A. and Milchev, G. (1974) Biochem. J. 143, 263--272 20 Perry, R.P. and Kelley, DoE. (1972) J. Mol. Biol. 70, 265--279 21 Weinberg, R.A. and Penman, S. (1970) J. Mol. Biol. 47, 169--178 22 Maden, B.E.H. (1971) l~ogz. Biophys. Mol. Biol. 22, 127--176 23 Perry, R.P. and Kelley, D,E. (1968) J. Cell Physiol. 72, 235--245 24 Miller, L. (1973) J. Cell Biol. 59, 624--632
Biochimica et Biophysica Acta, 390 (1975) 319--326 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98295
EFFECT OF THE EXOTOXIN OF BACILLUS THURINGIENSIS ON THE BIOSYNTHESIS AND MATURATION OF MOUSE LIVER NUCLEAR RNA
VLADIMIR V. MACKEDONSKI
Institute of Biochemistry, Bulgarian Academy of Sciences, 13 Sofia (Bulgaria) (Received November 26th, 1974)
Summary The effect of the exotoxin of Bacillus thuringiensis on the in vivo incorporation of [14 C] orotic acid into mouse liver nuclear rRNA and low molecular weight RNA was studied. The following results were obtained. 1. The exotoxin does not inhibit the synthesis of 45 S pre-rRNA, but causes a breakdown of these molecules. 2. The exotoxin inhibits the conversion of 38 S pre-rRNA into 32 S and 21 S. 3. The exotoxin inhibits the labelling of nuclear 5 S RNA, whereas the labelling of 4.6 S pre-tRNA is not affected. It is suggested that 5 S RNA may control the processing of 45 S prerRNA.
Introduction Previously we have shown that the exotoxin of Bacillus thuringiensis strongly inhibits the synthesis of nuclear rRNA species and the labelling of cytoplasmic 28 S, 18 S and 5 S rRNA in mouse liver, whereas the labelling of heterogeneous nuclear RNA and 4 S RNA is affected only to about 50% [1,2]. Recent investigations with the exotoxin on partially purified DNA dependent RNA polymerases have yielded controversal results [3,4]. However, our studies on the inhibition of RNA polymerase activities in isolated mouse liver nuclei by the exotoxin have suggested, that the in vivo action of this compound could not be explained by inhibition of RNA polymerases [5]. In this paper we report experiments on the synthesis and maturation of nuclear rRNA and nuclear lmwRNA in the presence of low doses of exotoxin. Abbreviations: pre-rRNA and pze-tRNA, precursors of r R N A and t R N A , respectively, ImwRNA, l o w m o l e c u l a r weight RNA.
320 The results show that the exotoxin does not inhibit the synthesis of 45 S pre-rRNA, but causes a random breakdown of a part of these molecules. On the other hand, the exotoxin interferes with the early steps of rRNA maturation. The synthesis of nuclear 4.6 S pre-tRNA is not affected, but the synthesis of nuclear 5 S RNA is inhibited. Part of these results have been reported [6]. Materials and Methods
Condition of labelling in vivo. The experiments were carried out with albino mice weighing 20--25 g. Highly purified B. thuringiensis exotoxin was dissolved in 0.14 M NaC1 and injected i.p. 30 min (or in some experiments 120 min) before the introduction of 25 pCi per mouse of [2 -14 C] orotic acid (spec. act. 18.5 Ci/mol). Isolation of nuclei. Highly purified nuclei were obtained by the method of Blobel and Potter [7], modified as described earlier [8]. Isolation and fractionation of nuclear RNA. Nuclear RNA was extracted from highly purified nuclei by phenol treatment at different temperatures [1,9] as follows: the nuclear pellet was suspended in ice cold 1 : 1 (v/v) mixture of 0.14 M NaC1 and phenol. The mixture was homogenized briefly, shaken at 4°C for 15 min and centrifuged at 4000 X g in the cold. RNA in the aqueous layer was further deproteinized with phenol, containing 0.5% sodium dodecylsulfate, phenol/chloroform (1 : 1 by vol.) and chloroform. RNA in the final aqueous layer was precipitated with 2.5 vol. of ethanol, containing 1% potassium acetate. This fraction was further used for preparation of nuclear lmwRNA by precipitating the high molecular weight RNA with 2 M NaC1 for 12 h at --10°C. The interphase layer obtained after 4°C extraction was suspended in 0.14 M NaC1/phenol mixture (1 : 1 by vol.) and shaken for 15 min at 50°C. RNA extracted in the aqueous layer was deproteinized as described above. This fraction contained the bulk of the nucleolar RNA components and was designated as nuclear rRNA. In pulse labelling experiments nuclear rRNA was isolated from a crude nuclear pellet prepared by low speed centrifugation of 10% liver homogenate in 0.25 M sucrose, containing 10 mM MgC12, 25 mM KC1 and 50 mM Tris • HC1 (pH 7.8). The pellet was treated twice with phenol]NaC1 mixture at 4°C and nuclear rRNA was extracted for 15 min at 50°C. This was done because during the preparation of highly purified nuclei some maturation of rRNA occurs (Mackedonski, V., unpublished observation). Gel electrophoresis of R N A fractions. Agar gel electrophoresis and radioautography were performed by the method of Tsanev and Staynov [10] under conditions described earlier [1]. Since a simple correlation exists between the sedimentation coefficient of RNA and its mobility in agar gel [11] the separate components were designated further in the text by their approximate S values. In some experiments the dried agar gel plates were cut into 1 mm slices and counted directly in PPO/POPOP/toluene scintillant in a model 3320 Packard liquid scintillation spectrometer. The lmwRNA was analyzed by polyacrylamide gel electrophoresis [12]. Horizontal gel slabs (18 cm × 10 cm) of 10% polyacrylamide cross-linked with 0.5% N,N'-methylenebisacrylamide were used. The buffer was 18 mM Tris •
321
HC1 (pH 7.8), containing 15 mM NaH2P04 and 0.5 mM EDTA. The gel was pre-run for 60 min and RNA fractionation was carried out at 80--100 V for 6 h at 20°C. The gel slabs were stained overnight with "stains all" (Eastman Organic Chem.) [13]. The gels were cut into 1 mm slices, put into counting vials, incubated for 24 h at room temperature and then for 1 h at 60°C in a mixture of toluene/water/Protosol® (10 : 1 : 9, by vol.), cooled and counted in PPO/POPOP/toluene scintillant with efficiency for ' 4C of about 70%. Determination of radioactivity. 1--2 A: 60 units of RNA were precipitated in 5% trichloracetic acid in the presence of 2 A260 units carrier tRNA in the cold for 20 min. The precipitate was filtered through Whatman GF-C filters and counted in PPO/POPOP/toluene scintillant. Reagents. Analytical grade reagents were used throughout. The phenol was freshly distilled, saturated with 0.14 M NaC1 and contained 0.1% 8-hydroxyquinoline. HighIy purified exotoxin of B. thuringiensis was kindly supplied by Dr K. Sebesta, Institute of Organic Chemistry and Biochemistry, CAS, Praha. 2-[ 14C]Orotic acid was obtained from NAEC Institute for Isotopes, Budapest. Results
Dependence o f nuclear rRNA labelling on the dose of exotoxin. Fig. 1 shows that the inhibitory effect of B. thuringiensis exotoxin on the in vivo labelling of nuclear rRNA depends on the dose. The labelling of nuclear rRNA is suppressed to about 60% of the control values by a dose of 0.025 mg per mouse. Higher doses (0.20 mg per mouse) completely inhibit the nuclear rRNA labelling. Labelling o f 45 S pre-rRNA in the presence of exotoxin. The synthesis of 45 S pre-rRNA was studied in pulse labelling experiments. When mice are injected i.p. with [14 C] orotic acid for 15 rain the label is incorporated mainly in the fraction of 45 S pre-rRNA (Fig. 2A). Several faint, but clear cut peaks with mobilities corresponding to 38 S, 32 S and 24 S are also seen on the radioautograms. 100 L.
U c
60
'5 >
40
u ~
20
0
\ &
d.2 d3 d.4
05
mg exotoxin per mouse
Fig. I . I n h i b i t i o n o f in vivo [ 1 4 C ] o r o t i c a c i d i n c o r p o r a t i o n i n t o n u c l e a r r R N A o f m o u s e liver b y t h e e x o t o x i n o f Bac. thuringiensis. T h e m i c e w e r e i n j e c t e d i.p. w i t h v a r y i n g a m o u n t s o f e x o t o x i n 3 0 m i n b e f o r e t h e a d m i n i s t r a t i o n o f t h e l a b e l l e d p r e c u r s o r . 9 0 m i n labelling t i m e .
322 325 285 1.2-
~;24s
10-
]J 118S
E
A
C
45Sll
g 06 ~ O4
y
0.2 0
2
4
6
0 2 4 Mobility, cm from stort
6
0
4
6
8
Fig. 2. Agar gel electrophoresis of m o u s e liver nuclear r R N A of control (A) and exotoxin treated (B and C) animals. Mice were pretreated with 0.025 m g exotoxin per m o u s e for 30 (B) and 1 2 0 (C) rain and after that 25 #Ci per m o u s e of [14C] orotic acid was injected i.p. 15 rain labelling time, - - , absorbance, recorded at 2 6 0 n m , - ..... , radioactivity, recorded f r o m the blackening of radioautogzams at 5 5 0 n m .
When mice are pretreated with exotoxin for 30 or 120 min [14 C] orotic acid incorporation into the total nuclear rRNA is not suppressed (Table I). However, the electrophoretic analyses, followed by radioautography shows (Figs 2B and 2C) that the distribution of the labelled RNA for exotoxin pretreated animals differs from that of the controls. The radioactivity in the 45 S pre-rRNA peak strongly decreases (see also Table I, last column), while certain amount of heterogeneous labelled RNA appears moving ahead of 45 S prerRNA. It should be noted, however, that 24 S RNA still persists as a sharp peak. It may be suggested, therefore, that the overall effect of the exotoxin is not an inhibition of the synthesis of 45 S pre-rRNA but its degradation to fragments with lower molecular weight. S t u d i e s on r R N A maturation. Further we studied whether 45 S pre-rRNA breaks down completely or it is processed into its normal products to a certain extent. Mice were pretreated with exotoxin for 30 min and [14 C] orotic acid was injected for 30 and 120 min. In this case the incorporation of [14 C] oro-
TABLE I EFFE CT OF B A C I L L U S T H U R I N G I E N S I S EXOTOXIN ON THE PULSE LA B ELLIN G OF MOUSE LI VER NUCL E AR rRNA* Extoxin pretreatm e n t (rain)
Labelling
Incorporation (% o f c o n t r o l )
(rain)
Nuclear rRNA ( c p m / A 2 6 0 nm u n i t RNA)
0 30 120
15 15 15
1030 1020 958
I00 100 93
time
45 S pre-rRNA peak** cpm
inc orp oration (% of control)
620 310 260
I00 50 42
* Exotoxin in a dose of 0.025 rag/mouse was injected i.p. 30 or 1 2 0 rain before the administration of 25 ~Ci/mouse of [14C] orotic acid. ** T h e radioactivity of 45 S p r e - r R N A w a s determined in the following way: the peak of 45 S w a s located after the radioautogzaphy of the electzophoregzams, cut out and counted cllzect~y in toluene/ P P O / P O P O P phosphor.
323 T A B L E II E F F E C T O F B A C I L L U S T H U R I N G I E N S I S E X O T O X I N ON THE [ 1 4 C ] O R O T I C A C I D I N C O R P O R A T I O N IN M O U S E L I V E R N U C L E A R r R N A E x o t o x i n w a s administered i.p. 3 0 m i n b e f o r e [ 14C] orotic acid. Labelling time (min)
Radioactivity ( c p m / A 2 6 0 n m unit R N A ) Con~ol
Exotoxintreated
9430 13 5 3 5
5450 8987
30 120
Incorporation (% o f c o n t r o l )
57.8 66.4
rate into the total nuclear rRNA was inhibited to 60--65% of the controls (Table II). Nuclear rRNA was isolated from control and exotoxin-treated animals and was analyzed by agar gel electrophoresis. The A260nm profile of the nuclear rRNA shows the presence of 45 S, 32 S, 28 S, 21 S and 18 S peaks comparatively well separated (Figs 3A and 3B). Exotoxin pretreatment produces characteristic changes in the A260nm profile of nuclear rRNA (Figs 3C and 3D). The relative amounts of 45 S, 32 S and 21 S pre-rRNA peaks progressively decrease, compared to those of 28 S and 18 S rRNA peaks. Similar changes are observed with the newly synthesized RNA after both 30 and 120 min labelling. In controls the newly synthesized rRNA includes 45 S pre-rRNA and its normal maturation products, 32 S, 24 S and 21 S, after 30 min labelling and 32 S, 28 S, 21 S, 18 S and 15 S after 120 min labelling. 1.0
32S 28S
A
B
O.8 ~21S Q6
/,
0.4 E Q2 c O
0 L 1.0
C
D
0
fi = 0.8
0
"~ Q6 0.4 02 2
4 6 0 2 4 Mobility, cm from start
6
8
Fig. 3. Agar gel electxophoresis o f m o u s e liver nuclear r R N A o f c o n t r o l ( A and B) and e x o t o x i n treated (C and D) intmal~. Mice w e r e pretreated w i t h 0 . 0 2 5 m g e x o t o x i n per m o u s e and 3 0 m i n later [ 14C] o r o t i c acid w a s injected for 3 0 (A and C) and 1 2 0 (B and D) rain. - - , absorbance, r e c o r d e d at 2 6 0 n m , ...... , radioactivity, r e c o r d e d from the b l a c k e n i n g o f r a d i o a u t o g z a m s at 5 5 0 n m .
324
Q8 07 0.6 0.5 0.4 Q3 Q2 E c Q1 o
5S
o
~: 0.7 06 0.5 O4
4.6S
4S A
~20
c
!'
i',,
4o
E c
050 8
C
'200 '> '150
o.3!
100
0.2 I 01:
SO
.o "iJ
u
0
0
Direction of
run
Fig. 4. A c r y l a m i d e gel e l e c t r o p h o r e s i s o f m o u s e liver n u c l e a r I m w R N A f r o m c o n t r o l ( A and C) and e x o t o x i n t r e a t e d (B and D ) animals. Mice w e r e i n j e c t e d w i t h 0 . 0 2 5 m g e x o t o x i n per m o u s e and 3 0 m i n later [ 1 4 C ] o r o t i c acid w a s given i.p. for 3 0 ( A and B) and 1 2 0 (C and D ) min. - . . . . . , c h r o m o s c a n , r e c o r d e d at 5 5 0 n m ; - - - , radioactivity.
The same R N A species are presented also after pretreatment with exotoxin. However, characteristic changes are observed. The amount of radioactivity in 45 S, 32 S and 21 S rRNA peaks is markedly decreased as compared to that in 28 S and 18 S rRNA peaks. The labelling of 24 S R N A peak, however, is less (or is not) affected, as in the case of shorter labelling time (see Fig. 2). The results reveal that the 45 S pre-rRNA molecules synthesized in the presence of e x o t o x i n may undergo further processing, but at a slower rate, especially in the case of 38 S -* 32 S and 21 S conversion, as discussed below. Labelling of 5 S, 4.6 S and 4 S RNA. We have previously shown that higher doses of e x o t o x i n inhibited the labelling of 5 S rKNA almost completely, while the labelling of 4 S t R N A was less affected [ 1 , 6 ] . Here we confirm our observations with lower doses of exotoxin. We have found, that 5 S and 4.6 S RNA, the latter being identified as a precursor of t R N A [14,15] are more highly labelled than 4 S R N A is (Figs 4A and 4C). After treatment with e x o t o x i n for 30 (Figs 4A and 4B) or 120 min (Figs 4C and 4D) the specific radioactivity of 5 S R N A is about 50% of the control, while the labelling of 4.6 S R N A is unaltered. The labelling of 4 S R N A is only slightly affected (about 90% of the control). It should be noted that after 30 and 120 min labelling the incorporation of 14 C into 5 S R N A is inhibited to the same extent by the e x o t o x i n as that of 45 S pre-rRNA (see Fig. 2). Similarly, in our early experiments with higher doses of exotoxin, the labelling of 28 S and 18 S rRNA was found to be suppressed to the same degree as was the labelling of 5 S rRNA [1,6]. Discussion Our results have shown that the e x o t o x i n does not inhibit the synthesis of 45 S pre-rRNA but causes a rapid breakdown of a part of these molecules. The
325
45 S pre-rRNA molecules are synthesized in 2--5 min and have an average life time of about 15 min [16,17]. Consequently, in our pulse labelling experiments the radioactivity should be incorporated mostly into 45 S pre-rRNA. Since the low doses of exotoxin used in these experiments do not suppress the labelling of RNA (Table I) one could assume that the exotoxin does not inhibit the synthesis of 45 S pre-rRNA. On the other hand, the radioautograms show that in exotoxin treated animals 45 S pre-rRNA is much less labelled, while a heterogeneous population of RNA molecules with lower average molecular weight appears, indicating that most probably the exotoxin causes a rapid and. random degradation of a part of the 45 S pre-rRNA molecules. Another possibility is that the exotoxin removes the unterminated rRNA fragments from the DNA template, but it is less likely, because of the following reasons: 24 S RNA is believed to be the nonconservative part of pre-rRNA, located at the 3' end of the 45 S pre-rRNA molecule [18,19]. In the first step of rRNA processing 45 S pre-rRNA is split by an exonucleolytic scission into 38 S and 24 S RNA [20,21] (see the scheme). The presence of 24 S RNA peak even after 120 min exotoxin pretreatment strongly suggests that the exotoxin does not impede the synthesis of 45 S pre-rRNA. ~24S 45 S I)
I exotoxin
~ 32 S
~38S
, 28 S
lexoox
After exotoxin treatment the labelling of 32 S and 21 S pre-rRNA equals t h a t of 28 S and 18 S rRNA, respectively. This phenomenon could result either from an increase in the rate of 32 S and 21 S pre-rRNA maturation, or from a decrease in the rate of their formation. The first possibility is unlikely because, as we have previously shown, the labelling of cytoplasmic 28 S and 18 S rRNA species is suppressed to the same extent as is the labelling of the nuclear rRNA [1]. On the other hand, it is possible that the exotoxin inhibits the conversion of 45 S pre-rRNA into 38 S and 24 S. However, at 15 and 30 min labelling times (Figs 2 and 3C) the labelling of 24 S RNA is less (or is not) affected by the exotoxin as compared to that Of 32 S and 21 S pre-rRNA. Since 45 S pre-rRNA cleavage gives 24 S RNA and 38 S pre-rRNA, the latter being a common precursor of both 32 S and 21 S pre-rRNA (see the scheme), then these results indicate that the exotoxin interferes with the conversion of 38 S into 32 S and 21 S pre-rRNA. Studies on the nuclear lmwRNA have shown that 5 S and 4.6 S RNA have different sensitivity to the exotoxin. Since our results with alpha-amanitin [8] have shown that 5 S RNA is inhibited, while 4.6 S RNA is not, it may be suggested that 5 S and 4.6 S RNA are synthesized by different mechanisms (or polymerases). Finally, we should like to point out that the synthesis of nuclear 5 S RNA is inhibited to the same extent as is the labelling of 45 S pre-rRNA at both 30 and 120 min labelling. A similar correlation we observed with higher doses of exotoxin: the labelling of 28 S, 18 S and 5 S rRNA was inhibited to about 90%
326 (see ref. 1). Since the synthesis of 5 S RNA is not affected in the absence of rRNA synthesis and ribosome assembly [23,24] and since it is known that 5 S RNA is integrated into 80 S (or 55 S) ribosomal precursor ribonucleoprotein in the nucleolus [22], our results suggest, but do not prove, the possibility that 5 S RNA could control the turnover of pre-rRNA in the nucleolus. The normal processing of 80 S ribosomal precursor ribonucleoproteins in the nucleolus might depend on their proper assembly, so that the particles lacking 5 S RNA might be more susceptible to degradation. Acknowledgements I wish to thank Professor A.A. Hadjiolov for the helpful discussion, G.N. Dessev for the help with the manuscript and D. Kulekova for her excellent technical assistance. References 1 Mackedonski, V.V., Nikolaev, N., Sebesta, K. and Hadjiolov, A.A. (1972) Biochim. Biophys. Acta 272, 56--66 2 Maekedonski, V.V., Hadjiolov, A.A. and Sebesta, K. (1972) FEBS Lett. 21, 211--214 3 Smuckler, E.A. and Hadjiolov, A.A. (1972) Biochem. J. 129, 153--166 4 Beebee, T., Korner, A. and Bond, R.P. (1972) Bioehem. J. 127, 619--624 5 Maekedonski, V.V. and Hadjiolov, A.A. (1974) Dokl. Bolg. Acad. Nauk. 27, 1117--1120 6 Mackedonski, V.V. (1973) Ph.D. Thesis, Institute of Biochemistry, Sofia 7 Blobel, G. and Potter, V.R. (1966) Science 154, 1662--1665 8 Hadjiolov, A.A., Dabeva, M.D. and Mackedonski, V.V. (1974) Biochem, J. 138, 321--334 9 Markov, G.G. and Arion, V.J. (1973) Eur. J. Biochem. 33, 186--200 10 Tsanev, R.G. and Staynov, D.Z. (1964) Biokhimyia 27, 1126--1131 11 Hadjiolov, A.A., Venkov, P.V. and Tsanev, R.G. (1966) Anal. Biochem. 17, 263--267 12 Loening, U.E. (1970) Method Med. Res. 12, 359--368 13 Dahalberg, A.E.0 Dingman, C.W. and Peacock, A.C. (1969) J. Mol. Biol. 41, 139--152 14 Burdon, R.H. (1971) Progr. NucL Acid Res. Mol. Biol. 11, 33--79 15 Choe, B.K. and Taylor, M.N. (1972) Biochim. Biophys. Acta 272, 275--287 16 Chaudhuri, S. and Lieberman, I. (1968) J. Mol. Biol. 33, 323--326 17 Greenberg, H. and Penman, S. (1968) J, Mol. BioL 21, 527--536 18 Wallauer, P.K. and David, I.B. (1973) Proc. Natl. Acad. Sei. U.S. 70, 2827--2831 19 Hadjiolov, A.A. and Milchev, G. (1974) Biochem. J. 143, 263--272 20 Perry, R.P. and Kelley, DoE. (1972) J. Mol. Biol. 70, 265--279 21 Weinberg, R.A. and Penman, S. (1970) J. Mol. Biol. 47, 169--178 22 Maden, B.E.H. (1971) l~ogz. Biophys. Mol. Biol. 22, 127--176 23 Perry, R.P. and Kelley, D,E. (1968) J. Cell Physiol. 72, 235--245 24 Miller, L. (1973) J. Cell Biol. 59, 624--632
