Nucleic Acids Research, Vol. 20, No. 2 295-301

new rRNA cerevisiae

A

processing mutant of Saccharomyces

Lasse Lindahl, Richard H.Archer and Janice M.Zengel Department of Biology, University of Rochester, Rochester, NY 14627, USA Received September 16, 1991; Revised and Accepted December 16, 1991

ABSTRACT We have identified from a collection of temperature sensitive yeast mutants strains which fail to process rRNA normally. Characterization of one such mutant is reported here. This strain accumulates increased amounts of the 35S primary transcript, '24S' molecules extending from the transcription start site to the 5.8S region, and two classes of 5.8S rRNA with 5' extensions of 7 and 149 bases, respectively. We show that this pleiotropic change in the rRNA processing pattern is due to a single mutation. Possible models for the function of the mutated gene are discussed.

INTRODUCTION Ribosomal RNA in all organisms is transcribed as precursors which are processed concomitantly with their assembly together with ribosomal proteins (r-proteins) into functional ribosomes. The rRNA processing pathways in Saccharomyces cerevisiae are similar to those found in many other eukaryotic organisms. Thus, the 35S primary transcript (Fig. 1) contains the sequences of the mature 18S, 5.8S, and 25S rRNAs, flanked by 5' and 3' external transcribed spacers (ETS) and separated by internal transcribed spacers (ITS 1 and ITS2). After transcription has been completed, the 5' ETS sequence is removed from the 35S precursor rRNA (pre-rRNA) to form a 32S molecule. This is cleaved into 20S, 7S and 27S pre-rRNAs (Fig. 1) which are the precursors for 18S, 5.8S and 25S rRNA, respectively (reviewed in refs. 1, 2). Even though the major intermediates in this processing pathway were described almost two decades ago (3), our understanding of how the various spacers are removed and the relationship between these reactions and ribosomal assembly remains sketchy. Recent observations have implicated several types of small RNA molecules in rRNA processing. Direct involvement of a small nucleolar ribonucleoprotein particle (snoRP) containing U3 RNA is suggested by the observations that degradation of the U3 RNA or immune depletion of the U3 snoRP in an in vitro system derived from mouse prevents cleavage of the ETS of mouse rRNA (4). The importance of U3 is also indicated by the finding that disruption of Xenopus laevis U3 RNA in vivo blocks one of two alternate rRNA processing pathways (5). In addition, S. cerevisiae mutants lacking certain other small nuclear RNAs either process rRNA abnormally (6) or fail to produce mature 18S rRNA (7, 8). A role of snoRPs in rRNA processing is also implied by the finding that fibrillarin, a nucleolar protein associated with several snoRPs, is required for normal ribosome

formation (9). However, there is also evidence to suggest that rRNA processing and ribosome assembly may require more than the snoRPs. For example, deletions in certain S. cerevisiae r-protein genes which eliminate a polyubiquitin extension, normally co-translated with the r-protein moieties of these genes, lead to formation of unequal amounts of the two ribosomal subunits (10). A similar phenotype has been observed for a mutation in a gene which, according to its sequence motifs, encodes an RNA helicase (11). To further investigate rRNA processing, we have identified temperature sensitive S. cerevisiae mutants which fail to process rRNA normally. These mutants were found by screening a collection of about 1000 temperature sensitive strains (12). We are describing here one such strain (ts3at) containing a recessive mutation which results in multiple changes in the pattern of prerRNA. The abundance of 35S pre-rRNA is increased and 32S is absent, suggesting that an early event in rRNA processing might be affected by the mutation. Another very striking change observed in strains harboring the mutant gene is that a large fraction of the 5.8S rRNA has non-canonical 5' ends. Thus about half of the 5.8S rRNA molecules have a 7 base extension at the 5' end and about 10% have a 149 base extension. Finally, mutant strains accumulate a 24S pre-rRNA composed of the entire 5' ETS, 18S, ITS1, and the 5.8S rRNA sequence. Possible explanations for the pleiotropic nature of this mutation are discussed.

MATERIALS AND METHODS Strains and media Strains SS330 (MATa, ade2-101, his3A200, ura3-52, tyrl) and SS328 (MATa, ade2-101, his3A200, ura3-52, lys2) were obtained from J. Abelson (Caltech) and were used as wild-type strains. A collection of temperature sensitive mutants generated from SS330 and SS328 by Vijayraghavan et al. (12) was screened for potential rRNA processing mutants as described in the Results section. Cultures used for RNA preparations were grown in YPD medium (13).

RNA preparations Cultures were shaken at 250 until they reached an OD6w of about 0.8 as measured in a 10 mm cuvette in a Hitachi 124 spectrophotometer (about 107 cells per ml). Part of the culture was transferred to 370 and two hours later 0.3 ml samples of the cultures at each temperature were transferred to 2.2 ml

296 Nucleic Acids Research, Vol. 20, No. 2 microcentrifuge tubes which had been prewarmed to 900 and contained 0.3 g glass beads (Sigma G-9143; acid washed), 0.3 ml TSEI-SDS (0.02 M Tris-HCl, pH 7.4; 0.2 M NaCl; 0.04 M Na-EDTA; 0.1% sodium dodecylsulfate) and 0.6 ml watersaturated phenol. The tubes were then shaken on a Vortex mixer at top speed for five periods of 15 seconds interrupted with 30 second intervals of incubation at 900. The aqueous phase was further extracted with water-saturated phenol-CHCl3-isoamyl alcohol (25:24:1) and then with CHCl3-isoamyl alcohol (24:1). Finally, nucleic acids were ethanol precipitated and redissolved in TE (10 mM Tris-HCl, pH 7.4; 0.1 mM Na-EDTA). Northern analysis For analysis of large RNA molecules, 5 ,tg (0.1 A260 unit) aliquots of the RNA preparations were electrophoresed through denaturing agarose gels containing 6% formaldehyde essentially as described (14). The gel was stained with acridine orange (33 ,ug/ml in 0.01 M sodium phosphate, pH 6.8) and photographed, and the RNA was then blotted to a Hi-Bond N (Amersham, Inc.) membrane using the capillary transfer method (14). For analysis of RNA molecules shorter than about 400 bases, 5 ,ig aliquots of RNA were electrophoresed through 8% polyacrylamide-urea gels (160 mmX160 mmx0.3 mm) as described (15). The gel was stained with ethidium bromide (3 ,ug/ml) and photographed, and the RNA was transferred to a HiBond'membrane by electro-blotting in TAE buffer (10 mM TrisHCl, pH 7.8; 5 mM sodium acetate; 0.5 mM sodium EDTA) using a 50 V/cm gradient for 2.5 hrs. After blotting, the RNA was crosslinked to the membranes by exposing the moist blot to 254 nm light at 120 kJ/cm2 for 5 min in a Spectrolinker XL (Spectronics Corp.) and then baking at 800 for 2 hrs. The membranes were probed by hybridization in rotating chambers with the indicated radioactive DNA molecules. For probes prepared from restriction fragments, the hybridization was carried out for 24-48 hrs at 550 in a solution containing 50% formamide, 0.3 M NaCl, 0.02 M sodium phosphate (pH 6.8), 2.5x Denhardt's solution (ref. 16; 5' -3', Inc.), 10% polyethylene glycol 6000, 1% sodium dodecylsulfate, and 12 1g/ml sheared salmon sperm DNA (5' - 3', Inc.). Alternatively, the hybridization was performed for 2 hrs at 650 in Rapid Hybridization Buffer (RPN 1518; Amersham, Inc.). After hybridization the blots were washed as described (17). For probes prepared from oligonucleotides, the hybridization was carried out for 3-4 hrs at 370 in the sodium buffered solution described above, except that no formamide was used. The membranes were washed at room temperature in the solutions described by Hurley et al. (17) for a total time of about 10 min. Hybridization probes 32P-labeled ITS1 and ITS2 hybridization probes were prepared from purified restriction fragments by nick-translation or randomprimer extension essentially as described (14). An ITSl fragment was prepared from M13 phage rD4, obtained from J.R. Warner (Albert Einstein College of Medicine), and contains the promoterdistal eight bases from the 18S rRNA gene, all of ITS 1 and the 19 promoter-proximal bases from the 5.8S rRNA gene (Fig. 1). An ITS2 fragment was prepared from plasmid pDK2. This plasmid was constructed by cloning into pGEMZ7f(+) (Promega, Inc.) a SphI-ClaI fragment containing the 23 promoter-distal bases of the 5.8S gene and the 194 promoter-proximal bases of ITS2 (Fig. 1). The source of this fragment was pSCRl which contains a single rRNA repeat unit of genomic yeast rRNA genes and was obtained from E.A. Morgan (Roswell Park Cancer Institute).

We also used as hybridization probes oligonucleotides complementary to the various regions of the rRNA transcript (Table 1 and Fig. 1). These oligonucleotides were synthesized by Oligos etc or by the Biotechnology Service Facility in the Department of Microbiology and Immunology at the University of Rochester Medical Center. Oligonucleotides were labeled with 32P by the polynucleotide kinase reaction or by the terminal transferase reaction essentially as described (14).

Primer extension Annealing of primers and primer extension at 370 and 60 mM KCl were performed as described previously (22).

RESULTS Identification of an rRNA processing mutant To identify yeast mutants which process rRNA abnormally we examined the precursor rRNA (pre-rRNA) in a collection of about 1000 temperature sensitive mutants established by Vijayraghavan et al. (12). These workers generously made available to us their Northern blots of total RNA prepared from each of these mutants 2 hrs after a shift from permissive (250) to non-permissive (370) temperature. To specifically visualize precursor rRNA on the blots we used as probes a mixture of DNA fragments from ITS1 and ITS2 (Fig. 1), since these sequences are found only in prerRNA and not in mature rRNA. Hybridization with a mixture of these probes revealed a number of mutants with abnormal patterns of pre-rRNA molecules. In the current study we have focused on a mutant called ts3ai. Probing with ITSI of Northern blots of RNA from this mutant revealed a novel band (i.e. not found in wild-type RNA) migrating slightly faster than 7S pre-rRNA on denaturing agarose gels. More detailed comparison of RNA extracted from mutant and wild-type cells growing at 25° or 370 showed that the ts3a mutant, in addition to the band in the 7S-region, also accumulated more than the wild-type of the 35S pre-rRNA as well as a band, called 24S pre-rRNA, migrating between the 20S and 27S pre-

_ __

* ~~~~35S1. 27S

27S Probes:

01190

8

ITS1 ITS2

18S

25S 5.8S

1 S| Probes:

09

ITS1

010

IS

011017 020

Figure 1. The Saccharomyces cerevisiae rRNA transcription unit. Center: Map of the rRNA transcript unit. Transcription is from left to right. The open boxes indicate mature rRNA sequences and the hatched boxes indicate regions which are excised and degraded during processing. Top: maps of major rRNA processing intermediates found in wild-type cells. Bottom: expanded map of Internal Transcribed Spacers. Vertical arrows indicate processing sites inferred from the mapping of ends of processing intermediates and mature rRNA species (18, 19). Hybridization probes used in this work are indicated by horizontal bars above the central map and below the enlarged map at bottom. ITS1 and ITS2 indicate restriction fragments (see Materials and Methods). 0-numbers indicate synthetic oligonucleotides complementary to the indicated portions of rRNA (see Table 1).

Nucleic Acids Research, Vol. 20, No. 2 297 rRNA bands (Fig. 2). As expected, the 35S pre-rRNA hybridized to both the ITS1 and ITS2 probes (Figs. 1 and 2). However, only the ITS1 probe hybridized to the 24S band and the band in the 7S region. Concomitantly with the increased abundance of these RNAs, the 32S precursor was reduced below the level of detection in the mutant. In several experiments we also observed a reduction in the abundance of the 20S and 27S prerRNAs in the ts3a mutant, but this difference was not as reproducible as the other changes in the pre-rRNA pattern described above. Interestingly, even though the ts3a mutant is temperature sensitive for growth, there was no significant difference in the pre-rRNA patterns observed at 25° and 37°. We will return to this observation below.

Genetic analysis of the ts3a mutant To determine if the altered rRNA processing and the temperature sensitive growth of ts3a were caused by a single mutation, we analyzed the transmission of the two characters in genetic crosses between mutant ts3a and the SS330 wild-type strain. Diploids resulting from such matings were temperature resistant and contained pre-rRNA indistinguishable from that of the wild-type (data not shown). Analysis of spores from the diploid strains suggested that the original ts3a mutant had at least two separate mutations generating a temperature sensitive phenotype for growth, since in some tetrads the temperature sensitivity/ temperature resistance ratio was 3: 1. However, the mutant preP-obe: Stmra!

ITS, ts3

Mapping of the unusual rRNA molecules in the mutant To map the mutant pre-rRNA molecules within the rRNA transcription unit, we probed the Northern blots of RNA from

TS2 ts3

t

rRNA pattern in five tetrads analyzed always segregated 2:2 and was always associated with temperature sensitive growth. The two temperature sensitive descendants with mutant prerRNA pattern obtained from an ascus with a 3:1 segregation of temperature sensitivity were used for a second set of matings with SS330. Analysis of 9 asci containing four viable spores (a large fraction of the asci contained non-viable spores) from one of these crosses showed that both temperature sensitive growth and the abnormal pre-rRNA pattern now co-segregated in a 2:2 pattern. Furthermore, all mutants exhibited all the changes in pre-rRNA accumulation, i.e. absence of 32S pre-rRNA, increased 35S and 24S pre-rRNAs, and the novel band in the 7S region of the gel (see also below). These genetic results suggest that we had crossed out from ts3cr a single mutation (or several closely linked mutations) causing all the changes in the pre-rRNA accumulation as well as temperature sensitive growth. No linkage was observed between this mutation and any of the other genetic markers of the strains used (ade2, ura3, his3, tyr], lys2). While our characterization of the ts3a mutant was in progress, we learned that another mutant, called rrp2, with the same phenotype had been isolated from a separate collection of temperature sensitive mutants by Shuai and Warner (23). We have exchanged mutant strains with Dr. Warner and found that our mutant and rrp2 fail to complement each other. It is thus probable that these two independently isolated mutations are located in the same gene; hence, we have designated our mutant as rrp2-2 (for Ribosomal RNA Processing).

TempiFD 24S

Table 1 203

S

Figure 2. Ribosomal rRNA precursor molecules in the ts3a mutant and in the wild-type parent. RNA was prepared from each strain growing at 250 and 37°. The RNA was analyzed by denaturing agarose gel electrophoresis and Northern blotting analysis using the ITS1 or ITS2 probes. 53 erd 0 fi

Probes:

TS1r

I;1

Probe

Complement positions

Region

01190 09 010 O11 017 020

23-44 24-48 201-225 326-350 24-48 134-158

5' ETS ITS1 ITS1 ITS 1 5.8S 5.8S

Positions are indicated in each portion of the primary transcript according to sequence data of Klootwijk et al. (ref. 20; 5' ETS), Veldman et al. (ref. 18; ITS1) and Rubin (ref. 21; 5.8S rRNA).

S?I,

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C). s F, rr

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o

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00(D

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=

w~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Is

5.8S A 5.8S

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Figure 3. Analysis of rRNA precursor molecules in the offspring from a cross between a temperature sensitive derivative of the ts3CI mutant and a wild-type strain (see text for detail). The 36 strains obtained from nine tetrads were grown at 250, shifted to 370 and RNA was prepared two hours later. The RNA was fractionated on denaturing agarose gels and blotted. The blots were probed sequentially with probes 01190, 09, 010 (results not shown), 011 and ITS2. Only results from two tetrads are shown.

W

r

1 W.v V1vW

Figure 4. Mapping of low molecular weight RNA in np2-2 and wild-type strains. RNA from cells incubated for 2 hrs at 370 was fractionated on a ureapolyacrylamide gel and blotted. Blots of sets of mutant (M) and wild-type (W) lanes from the same gel were probed with 011, 017 (Fig. 1, Table 1) or a mixture of the two oligonucleotides.

298 Nucleic Acids Research, Vol. 20, No. 2 offspring of the 9 asci described above with oligonucleotides specific for different regions of the rRNA transcription unit as well as with the ITS2 fragment (Fig. 1). The results from probing of RNA from the offspring of spores in two of the nine asci are shown in Fig. 3. The 24S band hybridized probe 01 190 from the 5' end of the 35S pre-rRNA and probes 09 and 011 from ITS 1 (Table 1; Fig. 3). It also hybridizes probes 017 and 020 from the 5.8S rRNA sequence (not shown), but does not hybridize the ITS2 probe (Fig. 3). These results suggest that the endpoints of the 24S molecule are at the transcription start site and the 3' end of 5.8S rRNA. The abnormal band in the '7S' region of the gel hybridized only to probe 011 (Fig. 1, Table 1) from the downstream part of ITS1 (Figs. 1 and 3). The RNA in this band was further characterized by blotting RNA separated on an 8 % ureapolyacrylamide gel. Probing of RNA from one of the rrp2-2 mutant strains with the 011 oligonucleotide revealed a single band not found with wild-type RNA (Fig. 4) which was about 310 nucleotides long (see also Fig. 5). Since this band did not hybridize to the 09 and 010 oligonucleotides (data not shown) specific for sequences in the upstream half of ITS1 (Fig. 1; Table 1), we surmised that the 310 nucleotide molecule had to contain part or all of the 158 nucleotide 5.8S rRNA. This was confirmed by showing that oligonucleotide 017, which is complementary to the promoter proximal region of 5.8S rRNA (Fig. 1, Table 1), also hybridizes to the 310 base RNA molecule (Fig. 4). We therefore call the 310 base transcript 5.8S B (Fig. 6). The 017 probe revealed two bands in the 5.8S region for both mutant and wild-type RNA (Fig. 4, see also Fig. 5). We will refer to the smaller form of 5.8S rRNA simply as 5.8S or canonical 5.8S and to the slightly longer form as 5.8S A rRNA. Comparison of the wild-type and mutant lanes in Fig. 4 suggests that the rrp2-2 mutation has a clear effect on the relative abundance of the 5.8S and 5.8S A rRNA. Based on inspection of the original autoradiograms from the experiments in Fig. 4 and 5 as well as several additional experiments, we estimate that the ratio of 5.8S:5.8S A:5.8S B rRNAs in rrp2-2 mutant strains is approximately 45:45:10. In wild-type cells the 5.8S A accounts for about 10% of the total 5.8S rRNA and 5.8S B is not detectable. To determine if the enhancement of the 5.8S A rRNA and the appearance of the 5.8S B rRNA cosegregated with temperature

sensitivity we used the 017 oligonucleotide to probe RNA from the 9 tetrads described above (Fig. 5). All temperature-sensitive offspring generated a 5.8S band, a strong 5.8S A band, and a 5.8S B band. All temperature-resistant offspring had the wildtype pattern with 5.8S and a weak 5.8S A band. Thus the analysis of the small pre-rRNAs supports our conclusion that the complex pattern of pre-rRNAs found in the ts3a mutant is due to a single mutation. The electrophoretic mobility (Fig. 5) indicated that the 5.8S B RNA has the same molecular weight, about 310 bases, as the normal 7S pre-rRNA. However, since the 5' end of the 7S prerRNA coincides with the 5' end of mature 5.8S rRNA (ref. 24; Fig. 1) while 5.8S B contains ITS1 sequences, the 5.8S B RNA must be distinct from 7S pre-rRNA. To confirm this we re-probed a Northern blot of one of the acrylamide gels with ITS2. A 7S pre-rRNA bands was visible for all progeny, both mutant and wild-type (Fig. 5). Thus even though 5.8S B and 7S co-migrate, they are clearly not identical.

Determination of 5' ends of 5.8S rRNA molecules The 5' ends of the 5.8S A and B rRNA molecules were mapped by primer extension using oligonucleotide 017 (Fig. 1, Table 1). Two clusters of bands appeared close to the 5' end of the 5.8S rRNA whether RNA from mutant or wild-type strains was used as template (Fig. 7a). The predominant bands in one of the these two clusters correspond to the 5' end of the canonical 5.8S rRNA (25), whereas the predominant band in the other cluster maps 7 bases closer to the promoter and corresponds to the 5' end of 5.8S A. Thus the 5.8S A rRNA appears to be identical to one of the two minor forms of 5.8S rRNA previously described (25; see also below). As expected the bands corresponding to the 5' end of the 5.8S A rRNA were relatively stronger in the mutant lanes than in the wild-type lanes. We do not know why reverse transcriptase generated clusters of bands rather than single bands. These clusters were also observed when oligonucleotide 020 was used as primer and were not converted to single bands by increasing the reaction

(b) [

_

m 18S

35S 24S

_1

25S

l

5.8S

(a) 1S

ITS1ITS2

(c) [

(d)

m

5.8S B :1 5.8S A

MAAUACAACACAC ....... 132 bases ..... UUUAAAAUAUUAAAAAC t 2 A tt 5.8S A5'end 5.8S 5'end L 20S aend 5.BS B5'end

Figure 6. Summary of rRNA processing in the rrp2-2 mutant. (a) Map of the

Figure 5. Analysis of low molecular weight rRNA in the offspring from the cross described in the legend to Fig. 3 and text. RNA was electrophoresed through a urea-polyacrylamide gel, blotted, and probed with 017 or ITS2. Results from three tetrads are shown (tetrads 1 and 2 are the same tetrads shown in Fig. 3). Radioactive molecular weight markers were prepared in transcription run-off reactions using T7 RNA polymerase (15).

rRNA transcription unit with expansion of the 5.8S rRNA region. Open and hatched boxes indicate regions which are preserved or degraded, respectively, during processing in the wild-type cells. Vertical arrows indicate wild-type processing sites inferred from mapping of endpoints of mature rRNA and rRNA processing intermediates (18, 19). (b) and (c) Maps of rRNA molecules whose concentration is increased in haploid strains containing the rrp2-2 mutant gene. (d) 5' endpoints of the 5.8S rRNA species described in the text. The 3' endpoint of the 20S rRNA (18) is also indicated for reference. The nucleotide sequence is from Veldman et al. (18).

Nucleic Acids Research, Vol. 20, No. 2 299 temperature to 450 and salt concentration to 120 mM. Published fingerprinting experiments demonstrated only one 5' end of canonical 5.8S rRNA, but did indicate the existence of minor amounts of 5.8S rRNA molecules with both 6 and 7 base extensions (25). However, our Northern blots with higher resolution showed only one band for each of the canonical and 5.8S A rRNAs (not shown). We therefore believe that the clusters of bands are an artifact of the reverse transcriptase reaction and that in our yeast strains the canonical 5.8S and the 5.8S A rRNA both have unique 5' ends. In addition to the bands already discussed, the analysis of the reverse transcription products from mutant RNA showed a single band 149 bases to the promoter proximal side of the 5' end of canonical 5.8S rRNA (Fig. 7). This band is probably due to the 5' end of the 5.8S B rRNA found only in the mutant, since the electrophoretic mobility of the 5.8S B indicates that this molecule is about 150 bases longer than the canonical 5.8S rRNA (Fig. 5). Thus the 5' end of the 5.8S B rRNA is just 4 bases from the 3' end of the 20S pre-rRNA (Fig. 6; ref. 18). The relatively weak band observed in the wild-type lane of Fig. 7b at the -149 position could be due to a small amount of 5.8S B rRNA in wildtype cells. However, since no 5.8S B band was observed in Northern analysis of wild-type RNA, it is more likely that the band in the wild-type lane of Fig. 7b is due to the 5' end of 27S pre-rRNA.

Isolation of revertants The genetic analysis described above suggested that the rrp2-2 mutation caused both temperature sensitive growth and abnormal rRNA processing. To test this view, we selected for temperature resistant revertants. We failed to obtain revertants by plating

rrp2-2 cells directly at 370, even after plating more than 1011 cells. However, when we selected at 330, we obtained colonies at a rate of about 10-6. Interestingly, 3 of the 23 revertants from the 330 plates that we tested also grew at 370, even though no revertants had resulted from plating directly at this temperature. In any case, Northern analysis indicated that the pattern of RNA hybridizing to ITS1 in all the revertants was very similar to the pattern in a wild-type strain (Fig. 8). The fact that temperature sensitive growth and abnormal rRNA processing co-revert supports the conclusion that both phenotypes are due to the same genetic lesion. We surmised that the colonies growing at 370 were probably true revertants, while the revertants which grew only at 330 might contain second site suppressor mutations. This hypothesis was confirmed by crossing revertants to a wild-type strain. When we crossed revertants which grew at 370, we did not obtain any temperature sensitive progeny, confirming that the ability to grow at 370 was due to a mutation closely linked to the original temperature sensitive mutation. In contrast, the temperature sensitive phenotype was readily recovered in crosses between wild-type and revertants which grew at 330 but not at 370. Analysis of rRNA from cells grown at 180 The experiments described above suggested that a single mutation is responsible for both the failure of rrp2-2 mutant cells to grow Strain: W R18R20 M R1 r- LO r LO SNM S S Tep ( Ln ,Temp. \co co C\jco co c\j co 35S = O" 32S -* 24S 20S -_ adwo 40 ..

R18 W R20 M uI)

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LC

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e

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Figure 8. Northern analysis of RNA from temperature resistant revertants isolated at 33°. RNA was isolated from revertants and an np2-2 mutant strain (M) growing at 250 and 37°. The RNA was fractionated on a denaturing agarose gel (a) or a urea-polyacrylamide gel (b). RNA was blotted from the gels and the blot was probed with the ITSl fragment (a) or the 017 oligonucleotide (b). Revertant R20 is viable at 370, whereas R18 and RI are viable at 330, but not at 370.

'..

Strain: WWMM WM n- coac T emp. Ln r-Ln co NCIO

4- A

c\u

=8

35S _ 32S 24S -_ 20S * -

s'.

i..:

MM WM cn ra o ca c

O

, 4- 5.8S B

5.8S A 5.8S

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Figure 7. Mapping of 5' ends of the 5.8S transcripts found in the rrp2-2 mutant. Oligonucleotide 017 was hybridized to total RNA from each of four strains from a single tetrad from the cross described in the legend to Fig. 3. The hybridized primers were extended with MLV reverse transcriptase and the products were analyzed by electrophoresis on a urea-polyacrylamide gel. Markers were obtained by dideoxy-nucleotide sequencing using 017 as primer and double-stranded pSCRI (see Materials and Methods) as template. Panels (a) and (b) are autoradiograms of lanes which were electrophoresed for different times. Pertinent bases are marked with base letters and superscripts indicating the position relative to the 5' base (A' l) of canonical 5.8S rRNA.

(.a)

(b)

Figure 9. Composition of rRNA precursor molecules in a rrp2-2 mutant strain growing at 18°. RNA was isolated from a wild-type and a mutant strain (from the cross described in Fig. 3) growing at 180, 25°, or 37°. The RNA was fractionated on a denaturing agarose gel (a) or a urea-polyacrylamide gel (b). RNA was blotted from the gels and the blot was probed with the ITSl fragment (a) or the 017 oligonucleotide (b).

300 Nucleic Acids Research, Vol. 20, No. 2 at 370 and the abnormal rRNA processing. However, since we observed little or no difference between the rRNA processing patterns at 250 and 370 in the mutant, we wondered if the abnormal rRNA processing was due to a temperature sensitive mutation. We therefore examined rRNA intermediates in wildtype and mutant cells grown at 180. As shown in Fig. 9, the pattern of bands hybridizing to ITS1 from the mutant was very similar to the wild-type pattern when the cells were grown at 180. Thus this experiment suggests that the abnormal processing of rRNA in the mutant is indeed temperature sensitive (see also Discussion).

DISCUSSION We have identified rRNA processing mutants in a collection of temperature sensitive strains of Saccharomyces cerevisiae. One such mutant, called ts3a, has been characterized and is described in this paper. By genetic crosses of ts3Ol with wild-type strains, we obtained strains containing a mutation, called rrp2-2, causing temperature sensitivity both of growth and of rRNA processing. However, these two functions do not exhibit the same temperature dependence. Thus rrp2-2 mutant strains can grow at temperatures up to about 300, but rRNA processing is abnormal already at 250. Nevertheless, genetic analysis strongly suggests that temperature sensitive growth and abnormal rRNA processing are caused by the same mutation. That is, the two phenotypes cosegregated in genetic crosses, and temperature resistant revertants also exhibited a normal pre-rRNA pattern. The bands observed in Northern analysis of RNA from rrp2-2 mutant strains differ from the wild-type pattern in several ways. These changes include increased abundance of the 35S primary transcript (we have not verified if the 3' external transcribed spacer is retained), a 24S RNA extending from the transcription start to the 3' end of 5.8S rRNA, and 5.8S molecules with extensions of 7 bases (5.8S A) and 149 bases (5.8S B) on the 5' end (Fig. 6). The rrp2-2 mutation is not affecting RNA processing in general, since mRNA splicing is normal in the ts3ca strain (U.M.Vijayraghavan and J.Abelson, personal communi-

cation). We have not investigated the fate of the 24S and the 'long' 5.8S rRNA molecules. They might be further processed into 'normal' rRNAs or they could be turned over. An interesting possibility is that they accumulate. In fact previous experiments (25) suggested that the 5.8S A rRNA in wild-type cells is metabolically stable and is incorporated into ribosomes. Our preliminary experiments (not shown) suggest that ribosomes containing 5.8S A rRNA are loaded onto polysomes in the mutant, although we do not know if ribosomes containing 5.8S A rRNA function normally in all respects. The existence of minor amounts of 5.8S rRNA similar to 5.8S A rRNA is not limited to yeast. Heterogeneous 5' ends of 5.8S rRNA have also been observed in Xenopus (26), mouse (27), and rat (28). The 5' end of the 5.8S B molecule may coincide with the 5' end of the 27S pre-rRNA (see above) and maps just four bases to the promoter distal side of the 3' end of the 20S pre-rRNA (Fig. 6; ref. 18). This suggests the 3' end of the 20S pre-rRNA and the 5' end of 5.8S B (and possibly the 5' end of 27S prerRNA) are not created by a single endonucleolytic cleavage event, but may, for example, require an exonuclease which removes a few bases from one or both molecules after the endonuclease reaction.

There are several ways in which the rrp2-2 mutation could cause the observed pleiotropic effect on rRNA processing. One possibility is that it alters a component required for a step in the normal rRNA processing. Such a step is likely to be at the beginning of the processing pathway, because of the increased abundance of the two pre-rRNA (35S and 24S) which have retained the externally transcribed spacer at the 5' end. This spacer is removed early in the wild-type cells (1,2). In this case the remaining rRNA processing nucleases might produce rRNA by non-conventional pathway(s) or by pathways which play only a minor role in wild-type cells. This model is similar to what has been observed in Escherichia coli lacking functional RNaseIII, the enzyme which normally excises the 16S and 23S rRNA domains from the primary rRNA transcript in this organism (reviewed in ref. 29). Processing of rRNA via an alternate pathway could lead to temperature sensitive growth, either because the pathway is efficient only at low temperature, or because it produces rRNA with somewhat different ends or different modification patterns, and ribosomes containing the altered rRNA may function at 250 but not at 37°. The latter possibility is consistent with the observation that about half of 5.8S rRNA molecules in the mutant are extended at the 5' end. Even though some 5.8S A rRNA is also found in wild-type cells, the strong amplification of the longer 5.8S molecules in the mutant could be detrimental at high temperature. A similar situation seems to exist in the E. coli RNase III mutant in which the alternate rRNA processing pathway leads to abnormal ends of the 23S (30). This mutant is temperature sensitive for growth, even though RNase III cleavage of pre-rRNA is blocked even at permissive temperature. The RRP2 gene need not necessarily code for an RNA processing enzyme or a component of a snoRP. Rather, the RRP2 product may affect the formation of normal processing substrates, for example by involvement in methylation of rRNA or the synthesis or nuclear import of ribosomal proteins. Finally, it is possible that the rrp2-2 mutation alters the relative rates of transcription of individual rRNA repeat units. If there are, perhaps just minor, differences in the sequences between individual rRNA repeat units, the transcripts from individual genes might be processed along separate pathways. Thus an altered processing pattern observed in the rrp2-2 mutant could be due to alterations in a factor controlling the use of different types of rRNA repeats, or to a change in the control of the relative number of copies of the various types of rRNA genes in wildtype and mutant cells. Heterogeneity in the sequence of the ITS has in fact been reported for humans (31, 32). We are not aware of evidence for heterogeneity in the transcribed rRNA sequences in S. cerevisiae, but a polymorphism of restriction enzyme sites in the non-transcribed spacer has been described (33).

ACKNOWLEDGMENTS We are grateful to U.Vijayraghavan and J.Abelson for making their strains and Northern blots available and E.A.Morgan for plasmids and stimulating discussions. We also thank J.R.Warner for the ITS1 probe, communicating unpublished results, and suggesting analyzing the RNA composition at 180, and E.Grayhack, E.Phyzicky and S.Consaul for answering numerous questions about yeast procedures. This work was supported by a research grant from the National Institute for Allergy and Infectious Diseases.

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