A 12 S precursor to 5.8 S rRNA associated with rat liver nucleolar 28 S rRNA

Biochimica et Biophysica Act~ 739 (1983) 79-84

79

Elsevier Biomedical Press

BBA91170

A 12 S P R E C U R S O R TO 5.8 S rRNA A S S O C I A T E D WITH RAT LIVER N U C L E O L A R 28 S rRNA K.P. D U D O V , K.V. HADJ1OLOVA, M.B. K E R M E K C H I E V , B.S. S T A N C H E V and A.A. H A D J I O L O V *

Department of Molecular Genetics, Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia (Bulgaria) (Received July 16th, 1982)

Key words: nucleolar RNA; RNA processing," 5.8 S RNA precursor," Ribosome biogenesis; RNA denaturation; (Rat liver)

The pre-rRNA and rRNA components of rat and mouse liver nucleolar RNA were analysed. It was shown that upon denaturation, part of the 32 S pre-rRNA is converted into 28 S rRNA and 12 S RNA. The 12 S R N A from mouse (Mr, 0.36"106) is larger than the one from rat (Mr, 0.32.106). The 12 S RNA chain is intact and resists denaturation treatment. The non-covalent binding of this RNA with nucleolar 28 S rRNA is stronger than that of 5.8 S rRNA with 28 S rRNA. Hybridization with a rat internal-transcribed spacer rDNA fragment identifies 12 S RNA as corresponding to the 5'-end non-conserved segment of 32 S pre-rRNA, including 5.8 S rRNA. The significance of the formation of a 12 S precursor to 5.8 S rRNA in the biogenesis of ribosomes in mammalian cells is discussed.

Introduction The molecule of 5.8 S r R N A seems to play an important role in the structure and biogenesis of eukaryotic ribosomes (reviews in Refs. 1 and 2). It is now firmly established [3,4] that the 5.8 S r R N A gene is located between the genes for S-rRNA and L - r R N A in the internal transcribed spacer (tS~). In the process of p r e - r R N A maturation in animal cells the 5.8 S r R N A sequences are present in 32 S pre-rRNA, the immediate nucleolar precursor of 28 S r R N A [5,6]. The molecular mass of 32 S p r e - r R N A exceeds by far the sum of the molecular masses of 28 and 5.8 S r R N A [7,8]. This fact indicates that more than one endonuclease cut of 32 S pre-rRNA is likely to be involved in the shaping of 5.8 S rRNA. Also, it suggests the possible existence of a precursor to 5.8 S r R N A containing additional tS~ sequences. In previous studies we have identified in mouse

* To whom correspondence should be addressed. Abbreviation: SSC, 0.15 M NaC1/15 m M sodium citrate. 0167-4781/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press

liver nucleolar R N A a distinct '15 S' R N A fraction [9,10]. Since upon 5-fluoroorotate block of 32 S pre-rRNA processing we found a parallel disappearance of label in both 28 S r R N a and 15 S RNA, we proposed that the latter molecule originates from non-conserved tS~ sequences in 32 S pre-rRNA [9]. More recently, a 7 S precursor to 5.8 S r R N A was identified in Saccharomyces carlsbergensis [11] and it was shown that it extends, by about 130 nucleotides, the Y-end of 5.8 S r R N A [12]. Larger precursors to 5.8 S r R N A were found also in HeLa and L cell nucleolar RNA, identified by fingerprint analysis [13] or by hybridization with appropriate r D N A restriction fragments [14]. In the present work we describe the identification in rat liver nucleolar R N A of a 12 S precursor to 5.8 S rRNA. The 12 S pre-rRNA molecule ( M r, 0.32.106) contains 5.8 S r R N A and practically all non-conserved sequences from the 5'-end of 32 S pre-rRNA. The 12 S pre-rRNA is non-covalently bound to nucleolar 28 S r R N A and this interaction is markedly stronger than that between 5.8 and 28 S rRNA.

80 Methods and Materials

Animals. The experiments were carried out with Wistar male albino rats (150 g) and male albino mice (20 g). R N A was labelled in vivo for 90 min by intraperitoneal administration of 20-50 ~Ci [~4C]orotate (specific radioactivity 37 m C i / m m o l ) per animal. Isolation of RNA. Pure liver nuclei were isolated by the two-step hyperosmotic sucrose/detergent method [8]. Nucleoplasmic and nucleolar r R N A were extracted by subsequent treatment of the nuclei with phenol at 4 and 50°C [15]. Cytoplasmic r R N A was extracted from the post-nuclear supernatant by treatment with phenol at 4°C [9]. Gel electrophoresis of RNA. The R N A was fractionated by electrophoresis in 1.5% agar/5 M urea slab gels at 5 V / c m for 4 h at 8°C [16]. The absorbance of the dried agar films at 254 nm was obtained by contact photoprinting and the radioactivity recorded by radioautography. Denaturation of RNA. In the denaturation experiments about 100 #g nucleoplasmic, nucleolar or cytoplasmic R N A were loaded on the gel. After electrophoresis, the bands of the separate prer R N A and r R N A components were visualized under ultraviolet light, cut out and processed further. In one group of experiments formamide was added to 50% ( v / v ) to the gel slices and R N A was denatured by heating for 5 rain at 70°C. Immediately after heating, the samples were loaded for re-electrophoresis in a g a r / u r e a gels as described above. In other experiments, the gel slices were forced through a syringe needle and the agar sedimented by centrifugation at 20000 x g for 30 min at 4°C. The supernatant was extracted twice with phenol in the cold and R N A precipitated with 2.5 vol. 96% ethanol/0.1 M sodium acetate (pH 5.0) at - 4 0 ° C for 30 min. The pellet was dissolved in buffer 1 (10 mM Tris-HCl (pH 8.0)/1 m M EDTA). Half of the sample was made up to 50% formamide and the R N A denatured as above. The other half was run in parallel gel slots as a reference for the purity of the individual R N A species tested. Hybridization with 3-'P-labelled rDNA. ~ Charon 4A bacteriophage D N A containing rat ribosomal R N A genes was kindly provided by Dr. V. Nosikov (Institute of Molecular Biology, Moscow).

Subcloning of different r D N A restriction fragments into pBR322 was carried out by standard methods [ 17]. In this study we used an EcoRI-BamHI r D N A fragment containing the T-end of the 18 S r R N A gene, the whole internal transcribed spacer (tS~) and about 1 / 4 of the 28 S r R N A gene. The recombinant plasmid containing this fragment is designated here as p8-rDNA. Endonuclease Sau3A subfragments of p8-rDNA (tS~-Sau3A) containing only internal transcribed spacer (see also Refs. 18 and 19) were recovered from standard agarose gels by the freeze-sqeeze method [20] (Fig. 1.). For hybridization experiments 0.5-1 /~g of p8r D N A or tS~-Sau3A r D N A were 32p-labelled by nick-translation in the presence of [a- 32P]dATP to a specific radioactivity of about 10 7 cpm per ~g D N A [21]. The hybridization of the electrophoretically fractionated nucleolar R N A with labelled D N A probes was carried out directly into the gel (Dudov, unpublished data). Briefly, about 10/~g of nucleolar RNA were fractionated by electrophoresis in 5 M urea/1.5% agar gel [16]. The gel was stained with ethidium bromide (0.5 /~g/ml), photographed, rinsed with several changes of 0.05% sodium dodecyl sulfate for 3-5 h at 10°C to remove urea and salts and dried under a stream of air. Prehybridization of the dried gel was for 1--3 h at 45°C in 50% ( v / v ) f o r m a m i d e / 5 x SSC/50 mM sodium phosphate (pH 6.5)/0.04% each of bovine serum albumin, Ficoll and polyvinylpyrrolidone. Labelled D N A probes (about 3.105 cpm) were added after heating at 100°C for 10 min. Hybridi-

ECOR/

"-~

~

~

8urn HI

5~s ]'[11

1

~b

~es I

A

I

~

I

A

k~

Fig. 1. Endonuclease Sau3A restriction map of the rDNA fragment used in hybridizationexperiments.The EcoRI-BamHI rat rDNA fragment was cloned in pBR322. The position of 5.8 S and portions of 18 and 28 S rRNA genes are indicated. The vertical lines designate Sau3A sites. The arrows indicate the fragments from the internal transcribed spacer used for hybridization. Due to their identical size these two fragments cannot be resolved by agarose gel electrophoresis. For a detailed restriction map of the EcoRI-BamHl rDNA fragment see Refs. 18 and 19.

81

zation was in the same buffer for 14-20 h at 45°C. It was followed by three 20 min washes with 5 × SSC/0.1% SDS at room temperature and three to five washes with 0.1 × SSC/0.1% SDS for 15 min each at 50°C. The gels were plated on a glass plate, dried on air (incompletely), covered with Saran wrap and exposed to X-ray film (without intensifying screen) for 24 h. Under the above conditions the low molecular weight 4-8 S RNA components are removed from the gel during the hybridization step. Results

In preliminary experiments we carried out comparative analyses of in vivo [J4C]orotate-labelled nucleolar RNA extracted from rat and mouse liver. The agar/urea gel electrophoresis (Fig. 2) reveals

a

b

c

the presence of all pre-rRNA and rRNA components identified previously [8,10,15]. In both mouse and rat liver nucleolar RNA a distinct fraction, located between 18 and 4 S RNA, is clearly seen. At different times of in vivo labelling, the label in this fraction increases simultaneously with that in known pre-rRNA (data not shown). It is noteworthy that this RNA fraction, isolated from mouse liver, migrates more slowly than the one from rat liver. Its molecular weight, as determined by acrylamide/formamide gel electrophoresis, is 0.32. l06 for the rat and 0.36.106 for the mouse. Accordingly, in agreement with others [14], we shall designate this nucleolar RNA fraction as 12 S RNA. Our previous studies provided indirect evidence that the 12 S RNA fraction in nucleolar RNA is related to pre-rRNA processing and originates from the internal nonconserved segment of pre-rRNA [9]. To elucidate further the origin of 12 S

d 0

45 41 36 32

S. S. SS

45 S 41 S I

32 S

28 S

28S'

21S 18 S

21 S

18S,

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b

c

d

e

f

g

........

41 S ~ _ ~ ~~' i 36 S-.,_ 32 S ~ 285~ 21 s ~ l l , , 18 s ~

12 S

12 S i~i~iii/i~i~il

12S~

4S~

Fig. 2. Differences in the electrophoretic mobility of pre-rRNA and r R N A components of nucleolar R N A from rat (a and b) and mouse (c and d) liver. Nucleolar R N A from rat and mouse liver (labelled in vivo with [14C]orotate for 90 min) were isolated as described in Methods and Materials. Fractionation by a g a r / u r e a gel electrophoresis was carried out according to Dudov et al. [16]. The samples were run in parallel with about 60 /~g R N A per gel slot. a and d, contact photoprints at 254 nm; b and c, autoradiograms. The positions of known pre-rR N A and r R N A are indicated, It is noteworthy that the 32 S and 12 S R N A fractions from rat and mouse differ in electrophoretic mobility.

Fig. 3. A g a r / u r e a gel electrophoresis of denaturated rat liver nucleolar pre-rRNA and rRNA. The preparation of t4Clabelled rat liver nucleolar R N A was the same as in Fig. 2. This nucleolar R N A was fractionated by agar-urea gel electrophoresis. The bands with the separate pre-rRNA and r R N A were cut out of the gel and the R N A was denatured by heating at 70°C for 5 min in 50% formamide. The samples were rerun in a g a r / u r e a gels under the same conditions. The autoradiograms of the separate pre-rRNA and r R N A are given: a, total nucleolar R N A ; b, 45 S; c, 41 S; d, 36 S; e, 32 S; f, 28 S, g, 21 S. The 18 S r R N A gives also a single band as 21 S pre-rRNA (not shown). Note that 12 S R N A is released only upon denaturation of 32 S pre-rRNA.

82

RNA we carried out denaturation experiments with the individual pre-rRNA species as separated by agar/urea gel electrophoresis. The results (Fig. 3) show that upon denaturation for 5 min, at 70°C in 50% formamide, all pre-rRNAs reveal the presence of hidden breaks, yielding high-molecular weight material with a heterogeneous distribution. The molecules of 18 S rRNA and its immediate precursor (21 S pre-rRNA) resist this denaturation treatment indicating the absence of hidden breaks [22]. As can be seen, this denaturation treatment releases a single distinct 12 S RNA fraction only from 32 S pre-rRNA. The association of 12 S RNA with the 32 S pre-rRNA fraction was analysed further by using a more highly labelled 32 S pre-rRNA recovered from the agar/urea gel in the cold. Under these conditions (Fig. 4) the denaturation of 32 S prerRNA yields only two major fractions comigrating with 28 S rRNA and 12 S RNA. We investigated also the release of 5.8 S rRNA from different

fractions of 28 S rRNA. As expected, the cytoplasmic and nucleoplasmic 28 S rRNA, isolated at 4°C, yield 5.8 S rRNA upon denaturation. In contrast, the 28 S rRNA from the nucleolar RNA fraction (isolated at 50°C) has totally lost its 5.8 S rRNA (Fig. 4). The split of 32 S pre-rRNA into 28 S rRNA and 12 S rRNA strongly suggests that the latter molecule corresponds to the non-conserved segment located at the 5'-end of 32 S pre-rRNA [3-6]. This possibility was investigated by hybridization of the nucleolar RNA components with the appropriate rat liver rDNA fragments. Hybridization with p8-rDNA yields a pattern identical with the one obtained with in vivo 14C-labelled nucleolar RNA (Fig. 5a). Hybridization with Sau3A tS:rDNA restriction fragments, correa

45S--~

b

c

.......... ~

d

~

36 S - - 4 1S ~ o

32S~ 28 S ~

12S~

b

c

d

e

f

32 S~

28 S 26 S ~ 21S

/

1

~'~"

~mlm

17 S 18 S ~ 12S~

~ -

- - - 5.8 S

4S--~

Fig. 4. Denaturation of rat liver 32 S pre-rRNA and 28 S rRNA. The R N A was labelled for 2 h in vivo with 50 btCi of [14C]orotate per animal. The 32S pre-rRNA and 12 S R N A were isolated and purified by a g a r / u r e a gel electrophoresis as described in Methods and Materials. The isolation of 28 S r R N A and its denaturation was as given in Fig. 3. Radioautography of the denatured R N A samples after re-electrophoresis in a g a r / u r e a gels: a, purified 32 S pre-rRNA before denaturation; b, 32 S pre-rRNA yielding 28 S and 12 S r R N A upon denaturation; c, 12 S RNA; d, cytoplasmic 28 S rRNA; e, nucleoplasmic 28 S rRNA; f, nucleolar 28 S rRNA. The samples in a - c and in d - f were run separately for different times and mobilities are not directly comparable. For details see text.

Fig. 5. Hybridization of rat liver pre-rRNA with 32p-labelled r D N A restriction fragments from the internal transcribed spacer. The total nucleolar R N A and 32 S pre-rRNA were isolated and purified as described in Methods and Materials. The analysis of the R N A samples was carried out by a g a r / u r e a gel electrophoresis [16]. The hybridization in the gel ( b - d ) was with 3. l0 5 c p m of nick-translated tSi-Sau3A r D N A fragments (see text and Fig. 1). a, nucleolar RNA, labelled in vivo with [t4C]orotate, showing the position of pre-rRNA and r R N A components; b - d , hybridization with 32p-labelled r D N A fragments; b, total nucleolar RNA; isolated 32 S pre-rRNA before (c) and after (d) denaturation.

83

sponding to the internal transcribed spacer (see Fig. 1), gives relevant signals in 32 S pre-rRNA and 12 S rRNA, while 28 S rRNA is negative (Fig. 5b). Experiments with isolated 32 S pre-rRNA (Fig. 5c and d) show directly that the tSi-rDNA fragments hybridize with 32 S pre-rRNA and 12 S rRNA, while the 28 S rRNA released upon denaturation is negative (see also Fig. 3b). These results identify, unambiguously, 12 S RNA as corresponding to the non-conserved 5'-end segment of 32 S pre-rRNA, where the 5.8 S rRNA sequence is located [5,6]. In addition, the hybridization experiments reveal (besides the known 45, 41, 36 and 21 S pre-rRNA) the presence in rat liver of three other RNA fragments, containing internal transcribed spacer sequences. Two of them may correspond to the respective 26 and 17 S pre-rRNA observed in mouse cells [14].

Discussion The results obtained in this work permit the identification in rat liver nucleolar RNA of a 12 S precursor to 5.8 S rRNA. This 12 S pre-rRNA is derived from the 5'-end of 32 S pre-rRNA and contains both 5.8 S and tS~ sequences. In fact, the estimated M r of 0.32. 10 6, indicates that 12 S prerRNA contains almost the complete non-conserved sequence of 32 S pre-rRNA (Mr, 2.15 • 10 6, see Ref. 8) plus the 5.8 S rRNA sequence located at or near its 5'-end [14]. This conclusion is supported further by the observation that in mice a larger 32 S pre-rRNA ( M r, 2.19-106, Ref. 26) releases a larger 12 S RNA ( M r, 0.36. 10 6) (Fig. 1). The observation of similar large precursors to 5.8 S rRNA in nuclei from mouse [9,10,14,23], rat [this work] and human [13] cells strongly suggests that their formation is a common feature of pre-rRNA processing in mammals. Accordingly, the most likely sequence in 32 S pre-rRNA processing involves two subsequent endonuclease cleavages: (1) 32 S pre-rRNA ---, 28 S rRNA + 12 S pre-rRNA (2) 12 S pre-rRNA ~ 5.8 S rRNA + nonconserved segment. It is noteworthy, that the processing of the immediate precursor to 28 S rRNA in mammals is apparently simpler than the one deduced from studies with Saccharomyces [11,24,25]. This may be

related to the existence of GC-rich double-stranded loops in the 5'-end segment of mammalian 32 S pre-rRNA [8,26-29]. Whether some 5'- or Y-end trimming of terminal nucleotides in 28 S rRNA is also taking place [30,31] remains to be ascertained. As shown here the 12 S pre-rRNA is tightly bound to 28 S rRNA and comigrates with the fraction of intact 32 S pre-rRNA molecules. Extraction under the same conditions (50°C) completely releases 5.8 S rRNA from either nucleolar [this work] or cytoplasmic [22,32] 28 S rRNA. Therefore, it is likely that non-conserved sequences in 12 S pre-rRNA contribute also to its interaction with 28 S rRNA. It is plausible that the strong interaction of 12 S pre-rRNA with nucleolar 28 S rRNA plays an important regulatory role in the formation of 5.8 S rRNA and the integration of late-adding structural ribosomal proteins into the large ribosomal subunit (see Ref. 2) and determines the observed stringent control [33] at this intranucleolar step of ribosome biogenesis.

Acknowledgment The authors are indebted to Mrs. Z. Stoymenova for her skillful technical assistance.

References 1 Wool, I.G. (1979) Annu. Rev. Biochem., 48, 719-754 2 Hadjiolov, A.A. (1980) in Subcellular Biochemistry (Roodyn, D.B., ed.), Vol. 7, pp. 1-80, Plenum press, New York 3 Speirs, J. and Birnstiel, M.L. (1974) J. Mol. Biol. 87, 237-256 4 Walker, T.A. and Pace, N.R. (1977) Nucleic Acids Res, 4, 595--601 5 Maden, B.E.H. and Robertson, J.S. (1974) J. Mol. Biol. 87, 227-235 6 Nazar, R.N., Owen, T.W., Sitz, T.O. and Busch, H. (1975) J. Biol. Chem. 250, 2475-2481 7 Hadjiolov, A.A. and Nikolaev, N. (1976) Progr. Biophys. Mol. Biol. 31, 95-144 8 Dabeva, M.D., Dudov, K.P., Hadjiolov, A.A., Emanuilov, I. and Todorov, B.N. (1976) Biochem. J. 160, 495-503 9 Hadjiolova, K.V., Golovinsky, E.V. and Hadjiolov, A.A. (1973) Biochim. Biophys. Acta 319, 373-382 10 Hadjiolov, A.A., Dabeva, M.D. and Mackedonski, V.V. (1974) Biochem..I. 138, 321-334 11 Trapman, J., DeJouge, P. and Planta, R.J. (1975) FEBS Lett. 57, 26-30 12 DeJonge, P., Kastelein, R.A. and Planta, R.J. (1978) Eur. J. Biochem. 83, 537-546 13 Khan, M.S.N. and Maden, B.E.H. (1976) FEBS Lett. 61, 10-13

84 14 Bowman, L.H., Rabin, B. and Schlessinger, D. (1981) Nucleic Acids Res. 9, 4951-4966 15 Dabeva, M.D., Dudov, K.P., Hadjiolov, A.A. and Stoykova, A.S. (1978) Biochem. J. 171,367-374 16 Dudov, K.P., Dabeva, M.D. and Hadjiolov, A.A. (1976) Anal. Biochem. 76, 250-258 17 Bolivar, F. and Beckman, K. (1979) Methods Enzymol. 68, 245-267 18 Rothblum, L.I., Parker, D.L. and Cassidy, B. (1982) Gene 17, 75-77 19 Subrahmanyam, C.S., Cassidy, B., Busch, H. and Rothblum, L.I. (1982) Nucleic Acids Res. 10, 3667-3680 20 Thuring, R.W.J., Sanders, J.P. and Borst, P. (1975) Anal. Biochem. 66, 213-220 21 Maniatis, T., Jeffrey, A. and Kleid, D.G. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1184-1188 22 Venkov, P.V. and Hadjiolov, A.A. (1969) Biochem. J. 115, 91-94 23 Hadjiolova, K.V., Naydenova, Z.G. and Hadjiolov, A.A. (1981) Biochem. Pharmacol. 30, 1861-1863

24 Helser, T.L. and McLaughlin, C.S. (1975) J. Biol. Chem. 250, 2003-2007 25 Veldman, G.M., Klootwijk, J., Van Heerikhuizen, H. and Planta, R.J. (1981) Nucleic Acids Res. 9, 4847-4862 26 Wellauer, P.K. and Dawid, I.B. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 2827-2831 27 Wellauer, P.K., Dawid, I.B., Kelley, D.E. and Perry, R.P. (1974) J. Mol. Biol. 89, 397-407 28 Schibler, U., Wyler, T. and Hagenbuchle. O. (1975) J. Mol. Biol. 94, 505-517 29 Wellauer, P.K. and Dawid, I.B. (1974) Brookhaven Symp. Biol. 26, 214-224 30 Hamada, H., Kominami, R. and Muramatsu, M. (1980) Nucleic Acids Res. 8, 889-903 31 Kominami, R., Hamada, H., Fujii-Kurijama, J. and Muramatsu, M. (1978) Biochemistry 17, 3965-3970 32 Pace, N.R., Walker, T.A. and Schroeder, E. (1977) Biochemistry 16, 5321-5328 33 Dudov, K.P., Dabeva, M.D., Hadjiolov, A.A. and Todorov, B.N. (1978) Biochem. J. 171,375-383