Molec. gen. Genet. 173, 203-210 (1979) © by Springer-Verlag 197~)

Saccharomyces cerevisiae Mutants Defective in the Maturation of Ribosomal RNA Pencho V. V e n k o v a n d A n g e l i n a P. Vasileva Department of Molecular Genetics, Institute of Molecular Biology, Bulgarian Academy of Scaences, 1113 Sofia, Bulgaria

Summary. Slow-growing m u t a n t s were isolated after mutagenesis of the osmotic-sensitive strain Saccharomyces cerevisiae V Y l l 6 0 . The isolated m u t a n t s in rich m e d i a have g e n e r a t i o n times f r o m 300 to 400 rain at 30 ° C. Studies o n the biosynthesis of r R N A x have shown, that the processing of 37S p r e - r R N A in 6 of the slow-growing m u t a n t s occurs 3 to 4 times slower t h a n in the p a r e n t a l strain. These m u t a n t s with decreased rate of r R N A m a t u r a t i o n are of two different types. In some of them the processing of both 37S a n d 27S p r e - r R N A is slowed down, while the m u t a n t s f r o m the second group are acharacterized by a specific i n h i b i t i o n of the step 27S prer R N A ~ 2 5 S r R N A . E x p e r i m e n t s in which the synthesis of m a c r o m o l e c u l e s was studied, have shown that in the m u t a n t s a n d in the p a r e n t a l strain, R N A a n d proteins are synthesized at c o m p a r a b l e rates. Prel i m i n a r y results suggest that the decreased rate of r R N A processing in three of the isolated m u t a n t s m i g h t be due to a n insufficient f u n c t i o n of the enzymes involved in the m a t u r a t i o n of r R N A .

Introduction In the yeast Saccharomyces cerevisiae the r i b o s o m a l R N A is t r a n s c r i b e d as a large precursor molecule the 37S p r e - r R N A , which c o n t a i n s the sequences of three of the four species of r R N A . The 37S prer R N A is cleaved e n d o n u c l e o l y t i c a l l y to give 27 S a n d 20S p r e - r R N A , which are the i m m e d i a t e precursors of the 25S a n d 18S r R N A ( U d e m a n d W a r n e r , 1972; T r a p m a n a n d Planta, 1976). The cleavage is catalysed by highly specific ribonucleases, b u t n o t h i n g is k n o w n

a b o u t the n a t u r e of the cleavage enzymes in eukaryotic cells. The available e x p e r i m e n t a l data strongly suggest that the biosynthesis of r R N A in e u k a r y o t i c cells is regulated m a i n l y post-transcriptionally, i.e. at the level of r R N A m a t u r a t i o n ( H a d j i o l o v a n d N i k o laev, 1976). Therefore, the isolation of m u t a n t s in the m a t u r a t i o n of r R N A m a y be helpful to u n d e r stand the c o n t r o l m e c h a n i s m which regulates the biosynthesis of r R N A . The p u r p o s e of the work we report here is to isolate a n d characterize yeast m u t a n t s defective in the m a t u r a t i o n of r R N A . Since the supply of 25S, 18S a n d 5.8S r R N A species limits the growth of yeast (Ludwig et al., 1977), we reasoned that such m u t a n t s are to be f o u n d a m o n g slow-growing yeast m u t a n t s an a s s u m p t i o n which torned out to be valid.

Materials and Methods Stra#ls and Growth Media. The osmotic-sensitivemutant Saccharomyees eerevisiae VY1160 was used. The strain VY1160 is a cytoplas-

mic deficient petite, temperature sensitive at 37°C and requires the presence of osmotic stabilizer in the medium (Venkov et al., 1974). The solid YPD medium and the low-phosphate and YM-5 media (Waltschewa et al., 1976) were supplemented with 10% sorbitol to support the growth of the osmotic-sensitive yeast ceils. The VYI160 cells are growing exponentially in these media with a generation time of 180 rain at 30° C. In some experiments, 3.2% NaC1 instead of i0% sorbitol was used as osmotic stabilizer. Under these conditions, the VY1160 strain is growing also exponentially, but with a generation time of 350 minutes at 30° C. Mutagenesis. The VY1160 cells were mutagenized with ethylmeth-

anesulfonate according to already described procedure (Sherman et al., 1970) except that in all buffers used, sorbitol (10% final concentration) was added to prevent the lysis of the fragile cells. Isolation and Analysis of RNA. Ribosomal RNA was extracted

For ofj))r#Tts contact: P. Venkov Abbreviations: rRNA to ribosomal RNA

ribosomal RNA; pre-rRNA

precursor

directly from the osmotic-sensitive cells as described previously (Venkov et al, 1977). RNA samples were analysed by electrophoresis in agar-urea gels (Dudov et al., 1976). The absorbance of the dried agar plates was recorded at 260 nm with a recording

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P.V. Venkov and A.P. Vasileva: S. cerevisiae Mutants Defective in the Maturation of Ribosomal RNA

spectrodensitometer. The plates were cut in 1 mm slices; RNA was hydrolysed with 5% NH4OH and the radioactivity counted with toluene-PPO-dimethyl-POPOP phosphor containing 30% Triton X 100 in a Packard Tricarb 3320 spectrometer. RNA samples labelled with 32p-orthophosphate or with 14C-uracil were analysed in the same way, but the dried agar plates were submited to autoradiography. Small molecular weight RNA species were analysed by electrophoresis in acrylamide-formamide gels as described by Staynov et al. (1972). Determiuation of the Relative Rates of RNA and Protein Synthesis. Cells were labelled in YM-5 medium with either 5 laC/ml of 3Huracil, or with 1 gC/ml of 14C-aminoacids mixure. The labelling has been made in presence of 5 ~tg/ml of cold uracil to assure a linear incorporation of the 3H-uracil into RNA for at least 3 h. The radioactivity insoluble in 5% TCA was determined in 1 ml culture samples (for measuring the protein synthesis the samples were heated at 90° C for 30 rain). The rates of RNA biosynthesis (R) and protein synthesis (P) of the mutants were determined according to Hartwell (1967) taking the rate of RNA and protein synthesis in the parental VY1160 strain for 1.00. In vitro Assay of the Maturation Activities. The maturation activities have been studied in cell-Iysates using pulse-labelled pre-rRNA as substrate. For obtaining cell-lysates, the osmotic-sensitive cells of VY1160 and the mutants isolated were lysed gently in destilled water (1/100 of the culture volume) for 1 min at 4 ° C. Alternatively the VYII60 cells were mechanically disrupted by grinding with glass beads in the cold. An aliquot of a 10 fold concentrated salt solution was added to give finai concentrations of MgCI2 5 raM, KC1 250raM, Tris-HC1 25 mM pH=7.6 (buffer 2). The lysates were clarified by centrifugation at 5,000 g for 20 min and the protein content determined in the supernatant by the method of Lowry et al. (1951). The ceIl-lysate thus obtained was used immediatelly. Usually 100 lag proteins and 200 lag rRNA were mixed in 200 lal final volume of buffer 2. After incubation at 15°C for 2 h, the RNA was purified by one deproteinization with phenol-1% SDS at room temperature, centrifuged and precipitated with ethanol. RNA samples were disolved in 6 M urea solution and aliquots containing 100 Itg RNA were subjected to electrophoresis in agarurea gels. For each preparation, a control sample containing RNA only, was incubated and analysed simultaneously with the experimental samples. Determb~ation of Ribonuclease Activities in Cell-Lysates. Total ribonuclease activities in yeast cell-iysates were determined according to the method of Ambellan and Hollander (1966), using highly labelled yeast rRNA as substrate. The reaction was stoped by the addition of La(NO3)3 in HC1 to a final concentration of 0.1 M and the radioactivity of RNA degradation products was determined in the 5,000 g supernatant. Materials'. Ethylmethanesulfonote is obtained from Eastman Organic Chemicals, New York. Nutritional media components are from Difco Laboratories, Michigan, USA. Radioactivelly labelled compounds: 3H-5,6-uracil (specific activity 58 mC/mM), 14C-2uracil (specific activity 61 mC/mM) and 14C-labelled amino acids mixure (specific activity 50 mC/mA) were obtained from The Radiochemical Centre, Amersham, U.K. ; Na2H32po4 carrierfree was from Isocommerz GmbH, GDR.

Results Isolation o f the Slow-Growing Mutants. T h e o s m o t i c sensitive s t r a i n S a c c h a r o m y c e s c e r e v i s i a e V Y 1 1 6 0 was

m u t a g e n i z e d w i t h e t h y l m e t h a n e s u l f o n a t e , t h e cells w e r e p l a t e d o n solid Y P D m e d i a a n d i n c u b a t e d at 30 ° C. W e d e f i n e d as s l o w - g r o w i n g m u t a n t s those, w h i c h f o r m e d w e l l - s h a p e d c o l o n i e s b e t w e e n the 7 th a n d t h e 14 th d a y o f i n c u b a t i o n . A m o n g 25000 c o l o n i e s tested, we selected 80 o f s u c h s l o w - g r o w i n g m u t a n t s . S i m i l a r l y to the p a r e n t a l V Y 1 1 6 0 strain, the i s o l a t e d m u t a n t s w e r e t e m p e r a t u r e sensitive at 37 ° C, s o r b i t o l d e p e n d e n t a n d o s m o t i c - s e n s i t i v e . W h e n s u s p e n d e d in b u f f e r s l a c k i n g the o s m o t i c stabilizer, the cells lysed and released between 60-80% of their cellular content. In rich m e d i a the m u t a n t s w e r e g r o w i n g at 30 ° C w i t h g e n e r a t i o n t i m e s v a r y i n g b e t w e e n 300 a n d 400 m i n u t e s . All m u t a n t s , f u r t h e r s t u d i e d in details, s h o w a l i n e a r g r o w t h rate, c o n t r a r y to the p a r e n t a l V Y 1 1 6 0 cells c h a r a c t e r i z e d by a m o r e r a p i d a n d e x p o n e n t i a l g r o w t h rate. Biosynthesis o f r R N A in the Slow-Growing Mutants. T h e f r a g i l i t y o f the i s o l a t e d m u t a n t cells facilitates the i s o l a t i o n o f u n d e g r a d e d R N A species a n d we s t u d i e d the s y n t h e s i s o f r R N A in the s l o w - g r o w i n g mutants. P r e l i m i n a r y e x p e r i m e n t s h a v e s h o w n t h a t in the p a r e n t a l V Y l l 6 0 cells l a b e l l e d f o r 20 rain w i t h 2 ~tC/ m l o f 3 2 p - o r t h o p h o s p h a t e in l o w - p h o s p h a t e m e d i u m , m o r e t h e n 9 5 % f r o m the r a d i o a c t i v i t y is a l r e a d y loc a t e d in the m a t u r e r R N A species. F o r s c r e e n i n g o f m u t a n t s w i t h d e c r e a s e d rate o f r R N A b i o s y n t h e s i s , the s l o w - g r o w i n g m u t a n t s w e r e l a b e l l e d w i t h 32p-ort h o p h o s p h a t e (2 g C / m l ) f o r l o n g e r p e r i o d s o f t i m e a n d r R N A i s o l a t e d a n d a n a l y s e d . W h e n the cells w e r e l a b e l l e d f o r 60 rain, a b o u t 3 0 4 0 % o f the r a d i o a c t i v i t y was f o u n d in p r e - r R N A f r a c t i o n s in 12 o u t o f 80 s l o w - g r o w i n g m u t a n t s tested. T h e s a m e results w e r e o b t a i n e d b y l a b e l l i n g the m u t a n t s w i t h 14C-uracil (0.1 g C / m l ) in Y M - 5 m e d i u m . A c c o r d i n g to the e l e c t r o p h o r e t i c p a t t e r n o f t h e i r r R N A , the m u t a n t s c o u l d be s e p a r a t e d in t w o differe n t g r o u p s . M o s t o f the 12 s l o w - g r o w i n g m u t a n t s h a v e e l e c t r o p h o r e t i c c h a r a c t e r i s t i c s s i m i l a r to t h a t s h o w n in Fig. 1 a i.e. an A260 r a t i o o f 2 5 S / 1 8 S = 1.8 f o r the p r e e x i s t i n g ( u n l a b e l l e d ) r R N A a n d a n a l m o s t e q u a l l a b e l l i n g o f 37S a n d 27S p r e - r R N A . In the m u t a n t s o f the s e c o n d g r o u p a r e d u c e d l a b e l l i n g o f t h e 25S r R N A w i t h a p r e f e r e n t i a l a c c u m u l a t i o n o f its i m m e d i a t e p r e c u r s o r , the 27S p r e - r R N A , was f o u n d (Fig. 1 b). T h e s e m u t a n t s are c h a r a c t e r i z e d also by a 25S/18S r a t i o o f a b o u t 1.2 f o r the u n l a b e l l e d r R N A . I n all s l o w - g r o w i n g m u t a n t s s t u d i e d the 20S p r e c u r s o r o f 18S r R N A d i s p l a y e d a r e l a t i v e l y l o w labelling. T h e e l e c t r o p h o r e s i s in a g a r - u r e a gels, u s e d f o r the a n a l y s i s o f the R N A s a m p l e s , is s u p p o s e d to give an a c c u r a t e e s t i m a t i o n f o r the m o l e c u l a r w e i g h t s o f

P.V. Venkov and A.P. Vasileva: S. cerevisiae Mutants Defective in the Maturation of Ribosomal R N A

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Mobitity in em from s t o r t Fig. l a and b. Electrophoresis in agar-urea gels of r R N A from the slow-growing mutants. VY55 (a) and VY34 (b) mutants were labelled with 0.1 gC/ml of 14C-uracil for 60 rnin, r R N A were isolated and analysed. The dried agar plates were used for autoradiography. Dotted lines absorbancy at 260 n m ; solid lines radioactivity recorded as absorbancy at 550 n m of the radiograms

the different pre-rRNA species (Dudov et al., 1976). We determined the molecular weights of the prer R N A fractions in the slow-growing mutants and found for 37S, 27S and 20S pre-rRNA species, values of 2.77x 106d, 1.58 x 106d and 0.83 x 106d respectively. The deviations in the molecular weight estimations found between the different mutant strains were within the limits of the errors of the method used. The values obtained for the molecular weights of the pre-rRNA species in the slow-growing mutants are very similar to those reported by other authors (Brand and Planta, 1975) and to the molecular weights of the corresponding R N A fractions in pulse-labelled wild types of yeast strains. In order to study the reasons for the decreased rate of r R N A biosynthesis, we carryied out pulsechase experiments with the 12 slow-growing mutants. The mutants were grown in YM-5 medium supplemented with 10% of sorbitol, while the parental V Y l l 6 0 cells were cultivated in the same medium but containing 3.2% NaC1 as osmotic stabilizer. Under these conditions, the generation times of all strains were between 350 and 400 minutes at 30 ° C. The cells were pulse labelled with 3H-uracil and then chased with cold uracil. The electrophoretic analysis of r R N A samples isolated before the chase (Fig. 2a, d, g) shows that 37S pre-rRNA is labelled predominantly. The chase with cold uracil was effective for 10 of the slow-growing mutants and for the VY1160 strain. In two mutants an increase of the specific radioactivities of r R N A preparations isolated after the chase was found, even when higher concentrations of cold uracil were used. The reasons for these straindependent differences were not clarified and the two

205

strains were omitted from further studies. The effectiveness of the chase in the remaining strains permits the study of the maturation of pre-synthesized 37S pre-rRNA. In the parental VY1160 cells, the 37S prer R N A is processed to mature r R N A 20 minutes after the chase (Fig. 2a, b, c), an observation very similar to that made by others (Udem and Warner, 1972). The same results we found in four of the 10 slowgrowing mutants studied. On the contrary, in 6 mutants a decrease in the maturation of 37S pre-rRNA was observed and 40 min after the chase a significant part of the radioactivity was still retained in the 37S and 27S pre-rRNA fractions. The processing of prer R N A in these mutants is completed between the 60 ~h and the 80 th minute after the chase. Fig. 2 presents the electrophoretic analysis of the processing of pulse-labelled 37S pre-rRNA in representatives from the two different groups of mutants. In mutants VY9, VY10 and VY55 the processing of both 37S and 27S pre-rRNA is slowed down (Fig. 2d, e, f). In the mutants V Y l l , VY33 and VY34, like in the parental strain, the 37S pre-rRNA disappears 20 min after the chase, but an inhibition of the step 27S p r e - r R N A ~ 2 5 S r R N A is observed (Fig. 2g, h, i). The 27S pre-rRNA accumulates in the cells and after incubation for longer periods of time, only part of the precursor is converted to the 25S rRNA. A 25S/18S ratio of 1.1 for the labelled r R N A was found 80 min after the chase in the VY11, VY33 and VY34 mutants, suggesting the degradation of a part from the 27S pre-rRNA within the mutant cells. The 27S pre-rRNA contains the sequences for the 5.8S r R N A (Udem and Warner, 1972) and the inhibition of the maturation of rRNA at the step 27S ~25S r R N A will cuse an inhibition of the synthesis of the 5.8S rRNA as well. On Fig. 3 the electrophoretic analysis of the small molecular weight R N A fractions from a pulse-chase experiments are presented. The radioactivity in the 5.8S r R N A appears 10-20 min after the chase in the parental VY1160 cells. On the contrary, in VY34 mutants the 5.8S region remains unlabelled till 40 rain after the chase, suggesting an inhibition of the 27S pre-rRNA maturation. Instead, low-molecular weight radioactive material appears after the chase in the region 7S-18S, probably because of the degradation of part of the 27S pre-rRNA molecules. These results indicate that in 6 of the mutants studied, the reason for the decreased biosynthesis of rRNA may be posttranscriptional and might involve some of the steps of rRNA maturation. The processing of the presynthesized 37S pre-rRNA in the mutants occurs 3 to 4 times slower when compared to the parental strain, suggesting a decreased rate of rRNA maturation in the mutants cells.

P.V. Venkov and A.P. Vasileva: S. cerevisiae Mutants Defective in the Maturation of Ribosomal RNA

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